Benzoxazepin oxazolidinone compounds and methods of use

ABSTRACT

Described herein are benzoxazepin oxazolidinone compounds with phosphoinositide-3 kinase (PI3K) modulation activity or function having the Formula I structure: 
     
       
         
         
             
             
         
       
         
         
           
             or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, and with the substituents and structural features described herein. Also described are pharmaceutical compositions and medicaments that include the Formula I compounds, as well as methods of using such PI3K modulators, alone and in combination with other therapeutic agents, for treating diseases or conditions that are mediated or dependent upon PI3K dysregulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) of U.S. Provisional Application Ser. No.62/188,029 filed on 2 Jul. 2015, which is incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to benzoxazepin oxazolidinone compoundswith activity against hyperproliferative disorders such as cancer. Theinvention also relates to methods of using the compounds for in vitro,in situ, and in vivo diagnosis or treatment of mammalian cells, orassociated pathological conditions.

BACKGROUND OF THE INVENTION

Upregulation of the phosphoinositide-3 kinase (PI3K)/Akt signalingpathway is a common feature in most cancers (Yuan and Cantley (2008)Oncogene 27:5497-510). Genetic deviations in the pathway have beendetected in many human cancers (Osaka et al (2004) Apoptosis 9:667-76)and act primarily to stimulate cell proliferation, migration andsurvival. Activation of the pathway occurs following activating pointmutations or amplifications of the PIK3CA gene encoding the p110α(alpha) PI3K isoforms (Hennessy et al (2005) Nat. Rev. Drug Discov.4:988-1004). Genetic deletion or loss of function mutations within thetumor suppressor PTEN, a phosphatase with opposing function to PI3K,also increases PI3K pathway signaling (Zhang and Yu (2010) Clin. CancerRes. 16:4325-30. These aberrations lead to increased downstreamsignaling through kinases such as Akt and mTOR and increased activity ofthe PI3K pathway has been proposed as a hallmark of resistance to cancertreatment (Opel et al (2007) Cancer Res. 67:735-45; Razis et al (2011)Breast Cancer Res. Treat. 128:447-56).

Phosphatidylinositol 3-Kinase (PI3K) is a major signaling node for keysurvival and growth signals for lymphomas and is opposed by the activityof the phosphatase PTEN. The phosphoinositide 3-dependent kinase (PI3K)signaling pathway is the most dysregulated pathway in hormone receptorpositive breast cancer (HR+BC). The PI3K pathway is also dysregulated inaggressive forms of lymphoma (Abubaker (2007) Leukemia 21:2368-2370).Eight percent of DLBCL (diffuse large B-cell lymphoma) cancers havePI3CA (phosphatidylinositol-3 kinase catalytic subunit alpha) missensemutations and 37% are PTEN negative by immunohistochemistry test.

Phosphatidylinositol is one of a number of phospholipids found in cellmembranes, and which participate in intracellular signal transduction.Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem. 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60). Phosphoinositide 3-kinases (PI3K) arelipid kinases that phosphorylate lipids at the 3-hydroxyl residue of aninositol ring (Whitman et al (1988) Nature, 332:664). The3-phosphorylated phospholipids (PIP3s) generated by PI3-kinases act assecond messengers recruiting kinases with lipid binding domains(including plekstrin homology (PH) regions), such as Akt and PDK1,phosphoinositide-dependent kinase-1 (Vivanco et al (2002) Nature Rev.Cancer 2:489; Phillips et al (1998) Cancer 83:41).

The PI3 kinase family comprises at least 15 different enzymessub-classified by structural homology and are divided into 3 classesbased on sequence homology and the product formed by enzyme catalysis.The class I PI3 kinases are composed of 2 subunits: a 110 kd catalyticsubunit and an 85 kd regulatory subunit. The regulatory subunits containSH2 domains and bind to tyrosine residues phosphorylated by growthfactor receptors with a tyrosine kinase activity or oncogene products,thereby inducing the PI3K activity of the p110 catalytic subunit whichphosphorylates its lipid substrate. Class I PI3 kinases are involved inimportant signal transduction events downstream of cytokines, integrins,growth factors and immunoreceptors, which suggests that control of thispathway may lead to important therapeutic effects such as modulatingcell proliferation and carcinogenesis. Class I PI3Ks can phosphorylatephosphatidylinositol (PI), phosphatidylinositol-4-phosphate, andphosphatidylinositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidylinositol-4-phosphate. Class III PI3Kscan only phosphorylate PI. A key PI3-kinase isoform in cancer is theClass I PI3-kinase, p110α as indicated by recurrent oncogenic mutationsin p110α (Samuels et al (2004) Science 304:554; U.S. Pat. No. 5,824,492;U.S. Pat. No. 5,846,824; U.S. Pat. No. 6,274,327). Other isoforms may beimportant in cancer and are also implicated in cardiovascular andimmune-inflammatory disease (Workman P (2004) Biochem Soc Trans32:393-396; Patel et al (2004) Proc. Am. Assoc. of Cancer Res. (AbstractLB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA; Ahmadi Kand Waterfield M D (2004) “Phosphoinositide 3-Kinase: Function andMechanisms” Encyclopedia of Biological Chemistry (Lennarz W J, Lane M Deds) Elsevier/Academic Press), Oncogenic mutations of p110α (alpha) havebeen found at a significant frequency in colon, breast, brain, liver,ovarian, gastric, lung, and head and neck solid tumors. About 35-40% ofhormone receptor positive (HR+) breast cancer tumors harbor a PIK3CAmutation. PTEN abnormalities are found in glioblastoma, melanoma,prostate, endometrial, ovarian, breast, lung, head and neck,hepatocellular, and thyroid cancers.

PI3 kinase (PI3K) is a heterodimer consisting of p85 and p110 subunits(Otsu et al (1991) Cell 65:91-104; Hiles et al (1992) Cell 70:419-29).Four distinct Class I PI3Ks have been identified, designated PI3K α(alpha), β (beta), δ (delta), and γ (gamma), each consisting of adistinct 110 kDa catalytic subunit and a regulatory subunit. Three ofthe catalytic subunits, i.e., p110 alpha, p110 beta and p110 delta, eachinteract with the same regulatory subunit, p85; whereas p110 gammainteracts with a distinct regulatory subunit, p101. The patterns ofexpression of each of these PI3Ks in human cells and tissues aredistinct. In each of the PI3K alpha, beta, and delta subtypes, the p85subunit acts to localize PI3 kinase to the plasma membrane by theinteraction of its SH2 domain with phosphorylated tyrosine residues(present in an appropriate sequence context) in target proteins (Ramehet al (1995) Cell, 83:821-30; Volinia et al (1992) Oncogene, 7:789-93).

The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such agents would be expected to inhibit cellularproliferation, to repress signals from stromal cells that provide forsurvival and chemoresistance of cancer cells, to reverse the repressionof apoptosis and surmount intrinsic resistance of cancer cells tocytotoxic agents. PI3K is activated through receptor tyrosine kinasesignaling as well as activating mutations in the p110 catalytic subunitof PI3K, loss of the tumor suppressor PTEN, or through rare activatingmutations in AKT.

Taselisib (GDC-0032, Roche RG7604, CAS Reg. No. 1282512-48-4, GenentechInc.), named as2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide,has potent PI3K activity (Ndubaku, C. O. et al (2013) J. Med. Chem.56:4597-4610; WO 2011/036280; U.S. Pat. No. 8,242,104; U.S. Pat. No.8,343,955) and is being studied in patients with locally advanced ormetastatic solid tumors. Taselisib (GDC-0032) is a beta-isoform sparinginhibitor of the PI3K catalytic subunit, 31× more selective for thealpha subunit, compared to beta. Taselisib displays greater selectivityfor mutant PI3Kα isoforms than wild-type PI3Kα (Olivero A G et al, AACR2013. Abstract DDT02-01). Taselisib is currently being developed as atreatment for patients with oestrogen receptor (ER)-positive,HER2-negative metastatic breast cancer (mBC) and non-small cell lungcancer (NSCLC). In the phase Ia study with single agent taselisib,partial responses (PRs) were observed in 6/34 enrolled patients. All 6responses were observed in patients with PIK3CA mutant tumors (Juric D.et al. AACR 2013), indicating the need to determine PIK3CA mutationstatus from patients treated with taselisib.

Recent clinical data with PI3K inhibitors has implicated PI3K deltaactivity as a source of gastrointestinal toxicities (Akinleye et alPhosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics”Journal of Hematology & Oncology 2013, 6:88-104; C. Saura et al “PhaseIb Study of the PI3K Inhibitor Taselisib (GDC-0032) in Combination withLetrozole in Patients with Hormone Receptor-Positive Advanced BreastCancer” San Antonio Breast Cancer Symposium—Dec. 12, 2014, PD5-2; Lopezet al “Taselisib, a selective inhibitor of PIK3CA, is highly effectiveon PIK3CA-mutated and HER2/neu amplified uterine serous carcinoma invitro and in vivo” (2014) Gynecologic Oncology).

Idelalisib (GS-1101, CAL-101, ZYDELIG®, Gilead Sciences Inc., CAS Reg.No. 870281-82-6,5-fluoro-3-phenyl-2-[(15)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone)is a selective PI3Kδ (delta) inhibitor and approved for the treatment ofchronic lymphoid leukemia, CLL (Somoza, J. R. et al (2015) J. Biol.Chem. 290:8439-8446; U.S. Pat. No. 6,800,620; U.S. Pat. No. 6,949,535;U.S. Pat. No. 8,138,195; U.S. Pat. No. 8,492,389; U.S. Pat. No.8,637,533; U.S. Pat. No. 8,865,730; U.S. Pat. No. 8,980,901; RE44599;RE44638). Diarrhea and colitis are among the most common adverse eventsreported after idelalisib treatment (Brown et al “Idelalisib, aninhibitor of phosphatidylinositol 3-kinase p110d, forrelapsed/refractory chronic lymphocytic leukemia” (2014) Blood123(22):3390-3397; Zydelig® Prescribing Information 2014; Zydelig® REMSFact Sheet). The significant GI toxicities observed after treatment withidelalisib are consistent with the hypothesis that inhibition of PI3Kδ(delta) is a source of gastrointestinal toxicities. Additional seriousside effects were seen in clinical trials of idelalisib (Zydelig®) incombination with other therapies. Adverse events, including deaths havebeen tied to infections such as pneumonia. In March 2016, the EMA'sPharmacovigilance Risk Assessment Committee (PRAC) issued a provisionalwarning and a recommendation that patients receive antibioticco-treatment and are routinely monitored for infection when takingZydelig (idelalisib). In March 2016, the US Food and Drug Administrationissued an alert that “six clinical trials exploring idelalisib(Zydelig®) in combination with other therapies have been halted due toreports of an increased rate of adverse events, including death”.

There is a need for additional modulators of PI3Kα that are useful fortreating cancers, particularly an inhibitor of PI3Kα that is selectivefor mutant PI3Kα expressing tumors relative to non-mutant PI3Kαexpressing cells. There is especially a need for such an agent thatselectively inhibits the PI3Kα isoform relative to the PI3Kβ, PI3Kδ, andPI3Kγ isoforms, which may be expected to result in an enhancedtherapeutic window.

SUMMARY OF THE INVENTION

The invention relates generally to benzoxazepin oxazolidinone compoundswith selective activity in modulating mutant forms of the PI3Kα (alpha)isoform, and having the Formula I structure:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof. The various substituents are defined herein.

Another aspect of the invention is a pharmaceutical compositioncomprising a benzoxazepin oxazolidinone compound of Formula I, and apharmaceutically acceptable carrier, glidant, diluent, or excipient.

Another aspect of the invention is a method of treating cancer in apatient having cancer comprising administering to said patient atherapeutically effective amount of a benzoxazepin oxazolidinonecompound of Formula I.

Another aspect of the invention is a kit for the therapeutic treatmentof breast cancer, comprising:

-   -   a) a benzoxazepin oxazolidinone compound of Formula I; and    -   b) instructions for use in the therapeutic treatment of breast        cancer

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the x-ray co-crystal structures of PI3Kα with taselisib(GDC-0032).

FIG. 1B shows the x-ray co-crystal structures of PI3Kα with Compound 529of U.S. Pat. No. 8,242,104.

FIG. 1C shows the x-ray co-crystal structures of PI3Kα with Compound101.

FIG. 1D shows the x-ray co-crystal structures of PI3Kα with Compound103.

FIG. 2A shows the x-ray co-crystal structures of PI3Kα with taselisib(GDC-0032).

FIG. 2B shows the x-ray co-crystal structures of PI3Kα with Compound101.

FIG. 3A shows Western-blot data depicting p110α(p110α, p110 alpha)levels after 24 hour treatment with Compound 101, Compound 103 andCompound 436 of U.S. Pat. No. 8,242,104 in HCC-1954 cells (PI3Kα mutantH1047R).

FIG. 3B shows Western-blot data depicting p110α(p110α, p110 alpha)levels after 24 hour treatment with Compound 101, Compound 103 andCompound 436 of U.S. Pat. No. 8,242,104 in HDQ-P1 cells (PI3Kαwild-type).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The nomenclature used in this Application is based on IUPACsystematic nomenclature, unless indicated otherwise.

DEFINITIONS

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo[4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo[5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro carbocyclyl moieties are also includedwithin the scope of this definition. Examples of spiro carbocyclylmoieties include [2.2]pentanyl, [2.3]hexanyl, and [2.4]heptanyl.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and thelike. Carbocyclyl groups are optionally substituted independently withone or more substituents described herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro heterocyclyl moieties are alsoincluded within the scope of this definition. Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl andazaspiro[2.4]heptanyl. Examples of a heterocyclic group wherein 2 ringatoms are substituted with oxo (═O) moieties are pyrimidinonyl and1,1-dioxo-thiomorpholinyl.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl.

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to slow down (lessen) an undesired physiologicalchange or disorder, such as the development or spread of arthritis orcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those with the conditionor disorder.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

“Hematological malignancies” (British spelling “Haematological”malignancies) are the types of cancer that affect blood, bone marrow,and lymph nodes. As the three are intimately connected through theimmune system, a disease affecting one of the three will often affectthe others as well: although lymphoma is a disease of the lymph nodes,it often spreads to the bone marrow, affecting the blood. Hematologicalmalignancies are malignant neoplasms (“cancer”), and they are generallytreated by specialists in hematology and/or oncology. In some centers“Hematology/oncology” is a single subspecialty of internal medicinewhile in others they are considered separate divisions (there are alsosurgical and radiation oncologists). Not all hematological disorders aremalignant (“cancerous”); these other blood conditions may also bemanaged by a hematologist. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. The myeloid cell line normally produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells; the lymphoidcell line produces B, T, NK and plasma cells. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Leukemias includeAcute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL) and small lymphocytic lymphoma (SLL).Lymphomas include Hodgkin's lymphomas (all four subtypes) andNon-Hodgkin's lymphomas (NHL, all subtypes).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: ibrutinib (IMBRUVICA™,APCI-32765, Pharmacyclics Inc./Janssen Biotech Inc.; CAS Reg. No.936563-96-1, U.S. Pat. No. 7,514,444), idelalisib (formerly CAL-101, GS1101, GS-1101, Gilead Sciences Inc.; CAS Reg. No. 1146702-54-6),erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®,Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS Reg. No.51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9,Pfizer), cisplatin (Platinol®, (SP-4-2)-diamminedichloroplatinum(II),cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CASNo. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®, CAS No.23214-92-8), Akti-1/2, HPPD, and rapamycin.

Chemotherapeutic agents include inhibitors of B-cell receptor targetssuch as BTK, Bcl-2 and JAK inhibitors.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate) and selective estrogen receptor modulators (SERDs) such asfulvestrant (FASLODEX®, Astra Zeneca); (ii) aromatase inhibitors thatinhibit the enzyme aromatase, which regulates estrogen production in theadrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane;Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA®(letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii)anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors,such as cobimetinib (WO 2007/044515); (v) lipid kinase inhibitors, suchas taselisib (GDC-0032, Genentech Inc.); (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, forexample, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, GentaInc.); (vii) ribozymes such as VEGF expression inhibitors (e.g.,ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as genetherapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®;PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®;ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab(AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (PERJETA™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), trastuzumab emtansine (KADCYLA®, Genentech Inc.), andtositumomab (BEXXAR, Corixia).

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a Formula I compound of this invention with a mammal for aperiod of time sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity. Enantiomers may be separated from a racemic mixture bya chiral separation method, such as supercritical fluid chromatography(SFC). Assignment of configuration at chiral centers in separatedenantiomers may be tentative, and depicted in Table 1 structures forillustrative purposes, while stereochemistry is definitivelyestablished, such as from x-ray crystallographic data.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts. The phrase “pharmaceuticallyacceptable” indicates that the substance or composition must becompatible chemically and/or toxicologically, with the other ingredientscomprising a formulation, and/or the mammal being treated therewith.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, aryl-aliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid “mesylate”, ethanesulfonic acid, p-toluenesulfonic acid, andsalicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and polyamine resins

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The term “EC₅₀” is the half maximal effective concentration” and denotesthe plasma concentration of a particular compound required for obtaining50% of the maximum of a particular effect in vivo.

The term “Ki” is the inhibition constant and denotes the absolutebinding affinity of a particular inhibitor to a receptor. It is measuredusing competition binding assays and is equal to the concentration wherethe particular inhibitor would occupy 50% of the receptors if nocompeting ligand (e.g. a radioligand) was present. Ki values can beconverted logarithmically to pKi values (−log Ki), in which highervalues indicate exponentially greater potency.

The term “IC₅₀” is the half maximal inhibitory concentration and denotesthe concentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed, and can be converted to an absolute inhibitionconstant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol.(1973) 22:3099). Other percent inhibition parameters, such as IC₇₀,IC₉₀, etc., may be calculated.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent hydrates, solvates, and polymorphs of suchcompounds, and mixtures thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent unlabeled forms as well as isotopicallylabeled forms of the compounds. Isotopically labeled compounds havestructures depicted by the formulas given herein except that one or moreatoms are replaced by an atom having a selected atomic mass or massnumber. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to 2H(deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S,36Cl, and 125I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labeled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labeled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent in the compound of the formula(I). The concentration of such a heavier isotope, specificallydeuterium, may be defined by an isotopic enrichment factor. In thecompounds of this invention any atom not specifically designated as aparticular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Accordingly, inthe compounds of this invention any atom specifically designated as adeuterium (D) is meant to represent deuterium.

Benzoxazepin Oxazolidinone Compounds

The present invention provides benzoxazepin oxazolidinone compounds ofFormula I, and pharmaceutical formulations thereof, which arepotentially useful in the treatment of cancer, having the structure:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof, wherein:

R¹ is selected from —CH₃, —CH₂CH₃, cyclopropyl, and cyclobutyl;

R² is selected from —CH₃, —CHF₂, —CH₂F, and —CF₃.

Exemplary embodiments of Formula I compounds include wherein R¹ is —CH₃.

Exemplary embodiments of Formula I compounds include wherein R¹ iscyclopropyl.

Exemplary embodiments of Formula I compounds include wherein R² is—CHF₂.

Exemplary embodiments of Formula I compounds include the compounds inTable 1.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. In some instances, the stereochemistryhas not been determined or has been provisionally assigned.

In addition, the present invention embraces all diastereomers, includingcis-trans (geometric) and conformational isomers. For example, if aFormula I compound incorporates a double bond or a fused ring, the cis-and trans-forms, as well as mixtures thereof, are embraced within thescope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

Biological Evaluation

The relative efficacies of Formula I compounds as inhibitors of anenzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. Determination of IC₅₀ values can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Exemplary Formula I compounds in Table 1 were made, characterized, andtested for binding to various isoforms and mutant forms of PI3Kaccording to the methods of this invention, and have the followingstructures, corresponding names (ChemBioDraw, Version 12.0.2,CambridgeSoft Corp., Cambridge Mass.), and biological activity. Wheremore than one name is associated with a Formula I compound orintermediate, the chemical structure shall define the compound.

TABLE 1 Formula I compounds No. Structure Name 101

(S)-2-((2-((S)-4-(difluoromethyl)- 2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9- yl)amino)propanamide 102

(S)-2-cyclobutyl-2-((2-((R)-4- methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9- yl)amino)acetamide 103

(S)-2-cyclopropyl-2-((2-((S)-4- (difluoromethyl)-2-oxooxazolidin-3-yl)-5,6- dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9-yl)amino)acetamide 104

(S)-2-cyclopropyl-2-((2-((R)-4- methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9- yl)amino)acetamide 105

(S)-2-cyclopropyl-2-((2-((S)-4- (fluoromethyl)-2-oxooxazolidin-3-yl)-5,6- dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9-yl)amino)acetamide 106

(S)-2-((2-((S)-4-(fluoromethyl)-2- oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9- yl)amino)propanamide 107

(S)-2-((2-((S)-4-(difluoromethyl)- 2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2- d][1,4]oxazepin-9- yl)amino)butanamide

Taselisib

The compound known as taselisib, GDC-0032, and Roche RG7604 (CAS Reg.No. 1282512-48-4, Genentech Inc.), has an IUPAC name:2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide,and the structure:

including stereoisomers, geometric isomers, tautomers, andpharmaceutically acceptable salts thereof.

Taselesib can be prepared and characterized as described in WO2011/036280, U.S. Pat. No. 8,242,104, and U.S. Pat. No. 8,343,955.

Pictilisib

The compound known as pictilisib, GDC-0941, Roche, RG-7321, andpictrelisib, (CAS Reg. No. 957054-30-7, Genentech Inc.,) is a potentmultitargeted class I (pan) inhibitor of PI3K isoforms. GDC-0941 iscurrently in phase II clinical trials for the treatment of advancedsolid tumors. GDC-0941 is named as4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine(U.S. Pat. No. 7,781,433; U.S. Pat. No. 7,750,002; Folkes et al (2008)Jour. of Med. Chem. 51(18):5522-5532), and has the structure:

including stereoisomers, geometric isomers, tautomers, andpharmaceutically acceptable salts thereof.

Alpelisib

The compound known as alpelisib (BYL719, Novartis, CAS#: 1217486-61-7)is an oral, selective inhibitor of the PI3K alpha isoform, and is inclinical trials for the potential treatment of a variety of tumor types,including a phase III study in combination with fulvestrant forsecond-line hormone receptor-positive, HER2-advanced metastatic breastcancer (Furet, P. et al (2013) Bioorg. Med. Chem. Lett. 23:3741-3748;U.S. Pat. No. 8,227,462; U.S. Pat. No. 8,476,268; U.S. Pat. No.8,710,085). Alpelisib is named as(S)—N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide)and has the structure:

Biochemical Inhibition of PI3K Isoforms

The ability of a compound of the invention to act as an inhibitor ofPI3Kα with selectivity over PI3Kβ, PI3Kδ, and PI3Kγ was determined usingthe methods of Example 901. The Ki values shown in Tables 2A and 2Brepresent the geometric mean of a minimum of three independentexperiments unless otherwise noted.

Table 2A shows the biochemical inhibition of four PI3K isoforms by theFormula I compounds of Table 1. In addition, two clinically tested PI3Kcompounds, taselisib and pictilisib are included as comparators. Therepresentative compounds of the invention exhibit strong activityagainst PI3Kα, and exhibit significantly enhanced selectivity relativeto the other isoforms PI3Kβ, PI3Kδ, and PI3Kγ when compared to taselisib(GDC-0032) and pictilisib (GDC-0941). In particular, the selectivityratios in the second from the right column of Table 2A show that eachFormula I compounds 101-107 has a PI3K alpha to delta selectivity ratiofar higher than taselisib or pictilisib. In fact, both taselisib andpictilisib have stronger activity against PI3K delta than against PI3Kalpha, i.e. their selectivity ratios are less than 1. The selectivityratios of Formula I compound 101-107 range from 301-fold to 634-fold.

Table 2B shows the biochemical inhibition of two PI3K isoforms, alphaand delta and the PI3K alpha to delta selectivity ratios for certaincomparator compounds of U.S. Pat. No. 8,242,104, and a compound bearinga dimethyloxazolidin-2-one group from U.S. Pat. No. 8,263,633 (Compound356, column 149). The comparator compounds shown here in Table 2B areexamples from the broad genuses described in each of U.S. Pat. No.8,242,104 and U.S. Pat. No. 8,263,633. Neither U.S. Pat. No. 8,242,104nor U.S. Pat. No. 8,263,633 disclose a compound within the scope of theFormula I compounds of the invention. While the representativecomparator examples of U.S. Pat. No. 8,242,104 as described in Table 2Bdemonstrate PI3Kα (alpha) vs. PI3Kδ (delta) selectivity ratios>1, themaximum observed selectivity ratio is 46.9-fold. Formula I compounds101-107 therefore achieve significantly higher selectivity ratios thanexamples of U.S. Pat. No. 8,242,104. There is no guidance in either U.S.Pat. No. 8,242,104 or U.S. Pat. No. 8,263,633 to make the selection ofstructural elements of the Formula I compounds to achieve the propertyof high PI3K alpha selectivity versus PI3K delta. This unexpectedproperty of greater than 300-fold PI3K alpha selectivity is conservedacross the entire spectrum of the compounds exemplified in Table 1.

Current PI3K inhibitors in clinical trials, such as taselisib (WO2011/036280; U.S. Pat. No. 8,242,104; U.S. Pat. No. 8,343,955), andother representative examples of U.S. Pat. No. 8,242,104 exhibitsignificant activity against the PI3Kδ (delta) isoform. This lack ofselectivity vs. PI3Kδ (delta) is consistent with observed GI toxicity inthe clinic for taselisib. There exists a need for inhibitors of PI3Kα(alpha) that contain the favorable characteristics representative ofexamples of U.S. Pat. No. 8,242,104 that are simultaneously lackingactivity against PI3Kδ (delta). The current invention provides compoundsthat meet this activity and selectivity profile.

The unexpected property of PI3K alpha selectivity is advantageous toremove gastrointestinal toxicity observed in clinical PI3K inhibitorcandidates. Recent clinical data with PI3K inhibitors has implicatedPI3K delta activity as a source of gastrointestinal toxicities (Akinleyeet al, “Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancertherapeutics” Journal of Hematology & Oncology 2013, 6:88-104). SeeTable 2 of PI3K inhibitors in clinical trials, taselisib and pictilisib.

With significantly higher selectivity for PI3Kα (alpha) inhibitionrelative to PI3Kδ (delta) inhibition, Formula I compounds 101-107 wouldtherefore be expected to achieve a greater margin between clinicalactivity driven by PI3Kα (alpha) inhibition relative to toxicitiesdriven by PI3Kδ (delta) inhibition, as compared to the clinically testedtaselisib and pictilisib or any of the examples of U.S. Pat. No.8,242,104 or U.S. Pat. No. 8,263,633. Accordingly, Formula I compoundsof the invention may be useful as therapeutic agents with a decreasedtoxicity profile relative to agents that exhibit greater inhibition ofthe normal functions of PI3Kβ, PI3Kδ, or PI3Kγ.

TABLE 2A Biochemical inhibition of PI3K isoforms by Formula I compoundsand comparator compounds taselisib and pictilisib SelectivitySelectivity Selectivity Compound PI3Kα Ki PI3Kβ Ki PI3Kδ Ki PI3Kγ Ki forPI3Kα for PI3Kα for PI3Kα No. (nM) (nM) (nM) (nM) vs. PI3K-β vs. PI3Kδvs. PI3Kγ taselisib 0.090 53.0 0.079 1.43 591 0.9 16.0 GDC-0032pictilisib 2.56 70.2 1.54 41.8 27.4 0.6 16.3 GDC-0941 101 0.034 99.712.2 18.2 2944 361 537 102 0.949 >1000 286 708 >1054 301 746 103 0.060335 37.7 49.0 5640 634 824 104 0.464 813 197 289 1750 425 622 105 0.051341 30.3 36.4 6718 598 717 106 0.040 122 16.7 17.8 3050 416 444 1070.048 132 17.5 23.7 2782 368 499

TABLE 2B Biochemical inhibition of PI3K isoforms by comparator compoundsSelectivity No. (U.S. Pat. PI3Kα Ki PI3Kδ Ki for PI3Kα No. 8,242,104)Structure (nM) (nM) vs. PI3Kδ 196 taselisib GDC-0032

  0.090  0.079  0.88 375

  0.016  0.417 21.7 436

  0.35  6.94 22.3 469

<0.02*  0.39* 19.3 486

  0.186**  2.5** 13.4 501

  3.56** 21.8**  6.1 529

  0.023  1.05 46.9 540

  2.72** 24.1**  8.8 544

  0.437** 11.3** 25.8 549 (separated stereoisomer 1)

  0.56**  9.41** 17.0 549 (separated stereoisomer 2)

  5.66**  1.70**  0.30 550

  0.107  1.96 19.4 356 (U.S. Pat. No. 8,263,633)

  0.967**  0.92**  0.95 *Ki value represents the average of twoexperiments, **Ki value represents a single experimentInteractions of Compounds with PI3K

A rational basis for PI3Kα selectivity by the Formula I compounds mayreside in certain binding interactions.

The ability of a compound of the invention to interact specifically withPI3Kα was determined by solving the x-ray co-crystal structure ofrepresentative compounds with PI3Kα (alpha) using the methods of Example908. Optimized structural design of PI3K inhibitors with selectivity forthe PI3Kα isoform over other isoforms may include precise positioningand arrangement of atoms and functional groups to interact withisoform-specific residues in the binding site. Particularly,substitution at the 9-position and at the 2-position of the5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine ring system are found tohave critical impacts on specific activity of compounds against PI3Kα.

FIGS. 1A-D show the x-ray co-crystal structures of taselisib (GDC-0032),reference compound 529 (U.S. Pat. No. 8,242,104) and two representativecompounds of the invention with PI3Kα. As shown in FIG. 1A, taselisib(GDC-0032) contains a primary amide functional group that is positionedwithin close contact of both Gln859 and Ser854, appearing to offer thepossibility of hydrogen-bonding interactions. The residue Gln859 isspecific to the PI3Kα isoform, with a different residue occupying thisposition in the other isoforms (PI3Kβ=Asp, PI3Kδ=Asn, PI3Kγ=Lys).However, despite this close contact with a PI3Kα-specific residue, asmeasured in a biochemical assay GDC-0032 has equal activity against bothisoforms PI3Kα and PI3Kδ, and only slightly reduced activity against theisoform PI3Kγ (see Table 2A).

As shown in FIG. 1B, reference compound 529 (U.S. Pat. No. 8,242,104)contains a primary amide functional group in a similar position to thatof taselisib. This functionality is within appropriate distance and ofappropriate geometry of both Ser854 and Gln859 to make hydrogen-bondinginteractions. The 46.9-fold selectivity ratio for PI3Kα relative toPI3Kδ (see Table 2B) may be rationalized in light of these interactions,and the knowledge that PI3Kδ does not contain a Gln residue at position859 and therefore these interactions should be specific to PI3Kα.

FIGS. 1C and 1D show that the primary amide of the(S)-2-aminopropanamide group of Compound 101 and the(S)-2-amino-2-cyclopropylacetamide group of Compound 103 each occupy avery similar place in the binding site to the primary amides of GDC-0032and reference compound 529 (U.S. Pat. No. 8,242,104). This primary amidefunctionality in each representative of the invention is withinappropriate distance and of appropriate geometry of both Ser854 andGln859 to make ideal hydrogen bonding interactions. Despite the apparentsimilarities in functional group placement and orientation, therepresentative examples illustrated in FIGS. 1C and 1D, as well as othercompounds of this invention with similar substituents and functionality,improve upon the interactions of the primary amide of both taselisib andreference compound 529 (U.S. Pat. No. 8,242,104) such that compounds ofthis invention are observed to have substantially increased selectivityfor PI3Kα relative to PI3Kδ as measured in a biochemical assay. Compound101 is 361-fold selective and Compound 103 is 634-fold selective, asubstantial increase relative to reference compound 529 (U.S. Pat. No.8,242,104) which is only 46.9-fold selective. In light of the similarityof the positioning of the primary amide functionality between taselisiband other compounds of U.S. Pat. No. 8,242,104 (as exemplified byreference compound 529), the increased selectivity for PI3Kα relative toPI3Kδ as demonstrated by compounds of this invention is an unexpectedproperty. There is no guidance in U.S. Pat. No. 8,242,104 to make theselection of structural elements of the Formula I compounds to achievethe property of high (>300-fold) PI3Kα selectivity. The Formula Icompounds of the invention improve upon the interactions of the primaryamide of GDC-0032 such that compounds of this invention are observed tohave substantially increased selectivity for PI3Kα relative to PI3Kδ asmeasured in a biochemical assay relative to comparator compounds (seeTables 2A and 2B).

FIG. 2A shows an x-ray structure of taselisib bound in the PI3Kα (alpha)active site. The N2 atom of the triazole ring is not able to interactdirectly with either the side-chain of Tyr836 (distance of 4.04 Å) orSer774 (distance of 2.74 and 2.82 Å, no complementary polarity betweenligand and residue). FIG. 2B shows an x-ray structure of Compound 101bound in the PI3Kα active site, and shows that the oxazolidinone ring isable to make multiple improved interactions with the protein relative tothe triazole ring. The carbonyl functionality is close to the Tyr836side chain (2.67 Å) and able to make a favorable polar interaction. Afluorine atom of the oxazolidinone substituent is in close contact (2.21Å) with the hydroxyl group of Ser774 and is consistent with a polarinteraction or non-classical hydrogen bond, a favorable interactionenabled by polarization of the carbon-fluorine bond (Böhm et. al,Fluorine in Medicinal Chemistry, (2004) ChemBioChem, 5:637-643; Zhou et.al, “Fluorine Bonding—How Does it Work In Protein-Ligand Interactions”,(2009) J. Chem. Inf. Model., 49:2344-2355).

All compounds of the invention contain an oxazolidinone ring and areable to make the improved interaction with Tyr836 of PI3Kα (alpha). Someexamples of the invention also contain a fluorinated substituent on theoxazolidinone ring and are able to make the improved interaction withSer774 of PI3Kα. Both of these binding interactions may contribute tothe enhanced selectivity for PI3Kα observed for examples of theinvention relative to examples of U.S. Pat. No. 8,242,104. As shown inTables 2A and 2B, compounds which contain the oxazolidinone ring havehigher isoform selectivity than comparable compounds which contain thetriazole ring. The residues Ser774 and Tyr836 are not unique to thePI3Kα isoform, PI3Kδ contains these same residues at the same positions,and the enhanced isoform selectivity of the oxazolidinone inhibitors isnot predicted by these crystal structures. Subtle differences in thepositioning and orientation of the same residue identity betweendifferent isoforms may result from subtle changes in secondary andtertiary protein structure. These differences are difficult to predictand interpret even in the face of x-ray crystal structures of bothprotein isoforms. The surprising and unexpected properties of improvedmolecular interactions and enhanced isoform selectivity of oxazolidinoneinhibitors is conserved across the entire spectrum of the compoundsexemplified in Table 1.

The oxazolidinone is structurally differentiated from the triazole inthat the oxazolidinone has a carbonyl, is more polar, and does not havearomatic character. The triazole does not have a carbonyl group, is lesspolar, and has aromatic character.

The oxazolidinone ring provides a further benefit relative to thetriazole ring in terms of increased sp3 character and reduced aromaticring count. It is generally accepted in the literature that an increasednumber of aromatic rings is correlated with an increased risk ofpromiscuous binding. By contrast, an increase in the fraction of sp3carbons (# sp3 carbons/# total carbons) is correlated with improvedphysicochemical properties and decreased promiscuous binding, decreasingthe risk of off-target toxicology. These concepts are described in thereferences Lovering et. al, “Escape From Flatland”, (2009) J. Med.Chem., 52:6752-6756 and Ritchie and Macdonald, “PhysicochemicalDescriptors of Aromatic Character and Their Use in Drug Discovery”,(2014) J. Med. Chem., 57:7206-7215. The replacement of the triazolearomatic ring as exemplified by representatives of U.S. Pat. No.8,242,104 with a saturated heterocyclic ring, the oxazolidinonecontained within every example of the invention, represents a favorabledecrease in the risk of off-target toxicology. The entirety ofexemplified compounds in U.S. Pat. No. 8,242,104 are overwhelminglypopulated by compounds with aromatic rings at this position, 4 examplesof a carboxamide functional group replacing the aromatic ring, and noexamples of saturated cyclic or heterocyclic systems. Due to thesignificantly different binding interactions and steric requirements ofaromatic and saturated heterocycles, they are typically notinterchangeable. With no examples of saturated heterocyclic systems atthe 2-position of the 5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepinering, U.S. Pat. No. 8,242,104 provides no guidance as to a method forreplacing the aromatic ring with a saturated heterocycle while retainingactivity against PI3Kα.

Accordingly, compounds of the invention contain optimized substituentsand functionality at both the5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine 9-position and2-position. These optimized compounds provide a significant andheretofore unknown benefit with respect to improved molecularinteractions and increased selective activity against PI3Kα, withreduced activity against PI3Kδ. The compounds of the invention may beuseful as therapeutic agents with an enhanced therapeutic windowrelative to related agents such as taselisib (GDC-0032).

Selective Inhibition of Mutant PI3Kα (Alpha)

The ability of a compound of the invention to act preferentially againstcells containing mutant PI3Kα was determined by measuring inhibition ofthe PI3K pathway in SW48 isogenic cell lines: PI3Kα wild-type(parental), helical domain mutant E545K, and kinase domain mutantH1047R, as described in the methods of Example 902.

Statistical Analysis: EC50 values represent the geometric mean of aminimum of 4 independent experiments unless otherwise noted. Allstatistics were performed using KaleidaGraph Software (version 4.1.3). AStudent t-Test was performed using unpaired data with equal variance tocompare activity against mutant cells and wild-type cells. P<0.05 isconsidered to be significant.

Table 3A shows the inhibition of P-PRAS40 in SW48 isogenic cells by theFormula I compounds of Table 1. These compounds all display increasedactivity against the mutant PI3Kα cells relative to the wild-type PI3Kαcells. Compounds of the invention show similar activity as taselisib inSW48 mutant PI3Kα cells, with equal or greater selectivity thantaselisib relative to activity in the wild-type PI3Kα cells (see Table3B).

Table 3B shows the inhibition of P-PRAS40 in SW48 isogenic cells bycertain comparator compounds of U.S. Pat. No. 8,242,104, a compoundbearing a dimethyloxazolidin-2-one group from U.S. Pat. No. 8,263,633(Compound 356, column 149), and pictilisib. The comparator compoundsshown here in Table 3B are examples from the broad genuses described ineach of U.S. Pat. No. 8,242,104 and U.S. Pat. No. 8,263,633. NeitherU.S. Pat. No. 8,242,104 nor U.S. Pat. No. 8,263,633 disclose a compoundwithin the scope of the Formula I compounds of the invention. Thecomparator compounds contain examples that do not have significantlyincreased activity for the mutant PI3Kα cells relative to the wild-typePI3Kα cells (see comparator compounds 436 and 549, p>0.05). Thesecompounds are very structurally similar to comparator compounds that doexhibit significantly increased activity for the mutant PI3Kα cellsrelative to the wild-type PI3Kα cell (see comparator compound 529).There is no common structural element within the comparator compoundsthat provides guidance towards selective inhibition of mutant PI3Kα(alpha) cells. More broadly, there is no guidance in either U.S. Pat.No. 8,242,104 or U.S. Pat. No. 8,263,633 to make the selection ofstructural elements of the Formula I compounds to achieve increased orequivalent activity against the mutant PI3Kα cells relative to thewild-type PI3Kα cells. This unexpected property is conserved across theentire spectrum of the compounds exemplified in Table 1.

TABLE 3A Inhibition of P-PRAS40 in SW48 isogenic cells by Formula Icompounds P-PRAS40 P-PRAS40 P-PRAS40 Fold Selectivity for FoldSelectivity for SW48(E545K) SW48(H1047R) SW48(parental) SW48(E545K) vs.SW48(H1047R) vs. No. EC50 (μM) EC50 (μM) EC50 (μM) SW48(parental)SW48(parental) 101 0.0027 0.0030 0.0063 2.4 (p < 0.001) 2.1 (p = 0.002)102 0.0335 0.0358 0.0849 2.5 (p = 0.002) 2.4 (p = 0.001) 103 0.00410.0038 0.0101 2.5 (p < 0.001) 2.6 (p < 0.001) 104 0.0200 0.0228 0.06183.1 (p = 0.007) 2.7 (p = 0.009) 105 0.0042 0.0048 0.0125 3.0 (p = 0.007)2.6 (p = 0.007) 106 0.0045 0.0044 0.0115 2.5 (p = 0.003) 2.6 (p = 0.001)107 0.0053 0.0052 0.0108 2.0 (p = 0.003) 2.1 (p = 0.003)

TABLE 3B Inhibition of P-PRAS40 in SW48 isogenic cells by comparatorcompounds Fold Selectivity Compound Fold Selectivity for or No. P-PRAS40P-PRAS40 P-PRAS40 for SW48(E545K) SW48(H1047R) (U.S. Pat. No.SW48(E545K) SW48(H1047R) SW48(parental) vs. vs. 8,242,104) EC50 (μM)EC50 (μM) EC50 (μM) SW48(parental) SW48(parental) 196 0.0034 0.00400.0079 2.3 (p < 0.001) 2.0 (p < 0.001) taselisib GDC-0032 pictilisib0.0284 0.0321 0.0315 1.1 (p = 0.7) 1.0 (p = 0.9) GDC-0941 436 0.00950.0092 0.0128 1.3 (p = 0.26) 1.4 (p = 0.22) GDC-0326 529 0.0008 0.00090.0016 2.2 (p < 0.001) 2.0 (p = 0.002) 549 0.0105* 0.0147* 0.0119* 1.10.8 (separated stereoisomer 1) *EC50 represents a single experiment

Antiproliferative Activity in PI3K Mutant Tumor Cells

The ability of a compound of the invention to act on PI3K mutant tumorcells was determined by measuring the cell viability EC50 in HCC1954cells (PI3Kα mutant H1047R) and MCF7 cells (PI3Kα mutant E545K) usingthe methods of Example 903. Table 4 shows that representative Formula Icompounds 101 and 103 of the invention are able to inhibit the PI3Kpathway and inhibit proliferation in HCC1954 cells and MCF7 cells with asimilar level of potency as comparator compounds taselisib (compound196, U.S. Pat. No. 8,242,104), pictilisib and compound 436 (U.S. Pat.No. 8,242,104).

TABLE 4 Anti-proliferative activity of PI3K compounds in mutantPI3K-alpha tumor cells HCC1954 antiproliferative MCF7 antiproliferativeCompound or No. EC50 (μM) EC50 (μM) Taselisib 0.04 0.02 196 (U.S. Pat.No. 8,242,104) GDC-0032 pictilisib 0.75 0.12 GDC-0941 436 (U.S. Pat. No.8,242,104) 0.35 0.09 101 0.06 0.03 103 0.07 0.03

In Vivo Efficacy

Tables 5-8 show data from in vivo tumor growth inhibition (TGI) studieswith PI3K compounds. Tumor volume change was measured for 20 days ormore in cohorts of immunocompromised mice bearing breast cancerxenografts, dosed daily by PO (oral) administration with vehicle andPI3K compounds (Example 904).

Table 5 shows that at maximum tolerated doses (MTD), GDC-0032(taselisib), Compound 103 and Compound 101 are each more efficaciousthan alpelisib (BYL-719) in a PI3K mutant tumor model.

Table 6 shows that at doses lower than maximum tolerated dose, (i.e. 25mg/kg), Compound 101 is more efficacious than GDC-0032 in a PI3K mutanttumor model. There is potential for greater therapeutic index (TI) withCompound 101 since maximum efficacy is reached before maximumtolerability.

Table 7 shows that at maximum tolerated doses, increased responses (PRsand CRs) are seen with Compound 101 compared to GDC-0032 in a PI3Kmutant tumor model. Also, at maximum tolerated doses, GDC-0032 andBYL-719 are equally efficacious.

Table 8 shows that at maximum tolerated doses, GDC-0032 and Compound 101are more efficacious than BYL-719, and Compound 101 is approximately asefficacious as GDC-0032 in a PI3K mutant tumor model.

TABLE 5 Comparison of maximum tolerated doses (MTD) of PI3K compounds inthe HCC-1954x1 (ER−, PI3K^(H1047R)) breast xenograft model PI3K Compound% TGI PR CR BYL-719, 40 mg/kg QD, PO 80 1 1 GDC-0032, 15 mg/kg QD, PO118 4 0 Compound 103, 100 mg/kg QD, PO 120 4 3 Compound 101, 50 mg/kgQD, PO 129 10 0

TABLE 6 Dose ranging study of Compound 101 in the HCC-1954x1 (ER−,PI3K^(H1047R)) breast xenograft model PI3K Compound % TGI PR CR Compound101, 0.5 mg/kg PO, QD 19 0 0 Compound 101, 1 mg/kg PO, QD 60 2 0Compound 101, 2.5 mg/kg PO, QD 68 1 0 Compound 101, 5 mg/kg PO, QD 84 00 Compound 101, 25 mg/kg PO, QD 140 7 0 Compound 101, 50 mg/kg PO, QD149 6 0 GDC-0032, 15 mg/kg PO, QD 111 2 0

TABLE 7 Dose ranging study of Compound 101 in the KPL-4 (ER−,PI3K^(H1047R)) breast xenograft model PI3K Compound % TGI PR CR Compound101, 1 mg/kg PO, QD 28 0 0 Compound 101, 2.5 mg/kg PO, QD 86 1 0Compound 101, 5 mg/kg PO, QD 93 1 0 Compound 101, 15 mg/kg PO, QD 125 50 Compound 101, 25 mg/kg PO, QD 135 9 0 Compound 101, 50 mg/kg PO, QD153 9 1 GDC-0032 15 mg/kg PO, QD 113 3 0

TABLE 8 Comparison of GDC-0032, Compound 101, and BYL-719 in the HCI-003(ER+, PI3K^(H1047R)) breast PDX xenograft model PI3K Compound % TGI PRCR BYL-719, 40 mg/kg PO, QD 114 2 0 GDC-0032, 15 mg/kg PO, QD 162 6 1Compound 101, 50 mg/kg PO, 175 5 2 QD

Pathway Inhibition in Isolated B-Cells

The ability of a compound of the invention to inhibit the PI3K-pathwayin B-cells was assessed by influence of the compounds on CD69 levelspost agonistic a-IgM treatment using the methods of Example 906. Theexpression of CD69 in B-cells resultant from a-IgM treatment is believedto be driven by signaling through PI3Kδ (delta). Table 9 showsrepresentative Formula 1 compounds are more selective inhibitors ofpathway signaling in a PI3K-mutant line (SW48 (H1047R)) vs. B-cells(column 3) compared to taselisib, pictilisib, alpelisib, compound-436(US U.S. Pat. No. 8,242,104) and idelalisib.

TABLE 9 Inhibition of CD69 expression in B-cells by select compounds.[B-cell a-IgM CD69 B-cell a-IgM CD69 expression IC50, expression IC50(μM), plasma-protein binding plasma-protein-bindingcorrected*]/[p-PRAS40 SW48 No. corrected* (H1047R) EC50] 101 0.047 16103 0.076 20 taselisib 0.00031 0.077 pictilisib 0.006 0.19 alpelisib0.048 0.79 Compound 436 0.020 2.2 (U.S. Pat. No. 8,242,104) idelalisib0.048 <0.048 *CD69 expression IC50 measured in human whole-blood andcorrected by multiplying by measured human f_(u) from a plasma proteinbinding experiment.

Pathway Inhibition in PI3K Mutant and Wild-Type Tumor Cells

The ability of a compound of the invention to inhibit PI3K-pathwaysignaling in tumor cells was assessed by measuring p-PRAS40 levels inHCC1954 (PI3Kα mutant H1047R) and HDQ-P1 (PI3Kα wild-type) lines, usingthe methods of Example 907. Table 10 shows representative Formula 1compounds 101, 103 and 105 are able to inhibit the PI3K pathwayselectively in PI3Kα mutant (HCC1954, PI3Kα mutant H1047R) vs. PI3Kαwild-type tumor cells (HDQ-P1, PI3Kα wild-type). Compounds 101, 103 and105 have greater mutant over wild-type selectivity than comparatorcompounds taselisib, pictilisib, alpelisib and compound-436 (U.S. Pat.No. 8,242,104).

TABLE 10 Inhibition of p-PRAS40 in HCC1954 and HDQ-P1 lines by selectcompounds. P-PRAS40 P-PRAS40 Fold Selectivity HCC1954 MSD HDQ-P1 MSD forHCC1954 No. EC50 (μM) EC50 (μM) vs. HDQ-P1 101 0.019 0.084 4.4 103 0.0280.155 5.5 105 0.027 0.179 6.6 taselisib 0.023 0.055 2.4 pictilisib 0.3240.094 0.3 alpelisib 0.483 0.311 0.6 Compound 436 0.089 0.122 1.4 (U.S.Pat. No. 8,242,104)p110α Degradation in PI3K Mutant Tumor Cells

The ability of a compound of the invention to decrease p110α levels wasdetermined in experiments with HCC1954 (PI3Kα mutant H1047R) and HDQ-P1(PI3Kα wild-type) lines, using the methods of Example 905. FIGS. 3A and3B show representative Formula 1 compounds 101 and 103 able to promotereduction of p110α levels selectively in PI3K mutant (HCC1954, PI3Kαmutant H1047R) vs. PI3Kα wild-type (HDQ-P1, PI3Kα wild-type) tumor cellsin a concentration dependent manner. FIG. 3A shows Western-blot datadepicting p110α(p110α, p110 alpha) levels after 24 hour treatment withCompound 101, Compound 103 and Compound 436 of U.S. Pat. No. 8,242,104in HCC-1954 cells (PI3Kα mutant H1047R). FIG. 3B shows Western-blot datadepicting p110α(p110α, p110 alpha) levels after 24 hour treatment withCompound 101, Compound 103 and Compound 436 of U.S. Pat. No. 8,242,104in HDQ-P1 cells (PI3Kα wild-type). Compounds 101 and 103 more stronglyinfluence p110α levels compared to compound-436 (U.S. Pat. No.8,242,104).

Multiple Day Oral Dosing in Dogs

The ability of a compound of the invention to promote gastrointestinaland/or systemic inflammation or cause lymphoid depletion was assessedvia clinical and anatomic pathology evaluation after multiple-day dosingin Beagle dogs (7-14 days). Formula 1 compounds 101 and 103 at >5-foldfree exposure multiples over TGI60 (tumor growth inhibition 60% in aPI3K-mutant xenograft study) do not promote a pro-inflammatory signatureas determined by clinical pathology or anatomic pathology evaluation(Table 11a, 11b). Similarly, compounds 101 and 103 produce only minoramounts of lymphoid depletion at high exposure multiples. In contrast,experiments with comparator compound taselisib indicate significantpro-inflammatory effects and lymphoid depletion at <0.3-fold freeexposure over TGI60 (Table 11c). Comparator compounds alpelisib(BYL-719) and compound 436 (U.S. Pat. No. 8,242,104) also causeinflammation and lymphoid depletion at lower exposure multiples comparedto Formula 1 compounds 101 and 103 (Table 11d, 11e). The extent andseverity of findings is consistent with increased inhibition of PI3Kδ(delta) at exposure multiples over CD69 IC₅₀ for the comparatorcompounds.

TABLE 11 Multi day dosing of Formula 1 and comparator compounds in dog.(a) Compound Exposure Exposure 101 AUC0- Cmax Exposure multiples Dose 24hr, D 14 D 14 Cmin multiples * to CD69 mg/kg/day (μM hr, (μM, D 14(total/free, IC50** Clinical Clinical Histopa- QD total/free)total/free) (μM, total) AUC) (total, C_(min)) Signs Pathology⁺ thology⁺0.05 0.23/0.16 0.033/0.023 0.0046 0.2x/0.6x 0.07x — — — 0.15 0.87/0.600.097/0.067 0.012 0.8x/2.3x 0.2x — — — 0.5  2.0/1.38 0.31/0.21 0.0171.9x/5.3x 0.3x — — — F_(u) dog = 0.692; F_(u) mouse = 0.252 (F_(u) =unbound fraction in species plasma) * Exposure multiples using TGI60from a 21-day KPL4 xenograft study; TGI60 @ 2 mg/kg, 1.04 μM hr total,0.26 μM hr free **a-IgM stimulated CD69 expression (whole-blood) IC₅₀ =67 nM ⁺Findings related to inflammation and lymphoid organs (b) CompoundExposure Exposure 103 AUC0- Cmax Exposure multiples Dose 24 hr, D 14 D14 Cmin multiples* to CD69 mg/kg/day (μM hr, (μM, D 14 (total/free,IC₅₀** Clinical Clinical Histopa- QD total/free) total/free) (μM, total)AUC) (total, C_(min)) Signs Pathology⁺ thology⁺ 0.1 0.27/0.14 0.04/0.020.0048  0.2x/4.7x 0.03x — — — 0.3  0.9/0.46  0.13/0.066 0.011 0.8x/15x0.08x — — — 1 2.54/1.29 0.31/0.16 0.034 2.2x/43x 0.2x Abnormal feces ↓Lymphoid (soft/mucoid, lymphocytes depletion: 4/4) thymus F_(u) dog =0.507; F_(u) mouse = 0.03 (F_(u) = unbound fraction in species plasma)*Exposure multiples using TGI60 from a 21-day HCC1954 TGI study; TGI60 @3 mg/kg, 1.13 μM hr total, 0.03 μM hr free **a-IgM stimulated CD69expression (whole-blood) IC₅₀ = 142 nM ⁺Findings related to inflammationand lymphoid organs (c) Exposure Exposure taselisib AUC0- Cmax Exposuremultiples Dose 24 hr, D 7 D 7 Cmin multiples* to CD69 mg/kg/day (μM hr,(μM, D 7 (total/free, IC₅₀ Clinical Clinical Histopa- QD total/free)total/free) (μM, total) AUC) (total, Cmin) Signs Pathology⁺ thology⁺ 0.30.44/0.12 0.07/0.02 0.002 0.1x/0.3x 0.7x — — Lymphoid depletion: lymphnodes, GALT, spleen, thymus GI inflammation: Stomach, neutrophilicinflammation 1  1.6/0.45 0.18/0.05 0.019 0.4x/1.0x 6.2x — — Lymphoiddepletion: lymph nodes, GALT, spleen, thymus GI inflammation: Dark redareas in stomach & rectum corresponding to inflammation in stomach 35.1/1.4 1.09/0.31 0.016 1.2x/3.2x 5.2x BW ↓ Lymphoid loss lymphocytesdepletion: (7.9% ↑ lymph nodes, vs pre-study; neutrophils GALT, spleen,primarily 1 dog) ↑ thymus thin, cool to monocytes GI touch (ears); ↑inflammation: abnormal globulins Dark red areas feces; emesis ↓ A:G instomach & ratio rectum ↑ corresponding fibrinogen to inflammation instomach, rectum, cecum Systemic inflammation: Neutrophil infiltrates inlymph nodes, spleen, thymus, liver, lung, kidney F_(u) dog = 0.28; F_(u)mouse = 0.10 (F_(u) = unbound fraction in species plasma) *Exposuremultiples using TGI60 from a 21-day KPL4 xenograft study; TGI60 @ 4.3 uMhr (total) or 0.44 uM hr free ** a-IgM stimulated CD69 expression(whole-blood) IC₅₀ = 3.1 nM ⁺Findings related to inflammation andlymphoid organs (d) Compound 436 (U.S. Pat. No. Exposure Exposure8,242,104) AUC0- Cmax Exposure multiples Dose 24 hr, D 7 D 7 Cminmultiples* to CD69 mg/kg/day (μM hr, (μM, D 7 (total/free, IC₅₀**Clinical Clinical Histopa- QD total/free) total/free) (μM, total) AUC)(total, Cmin) Signs Pathology⁺ thology⁺ 0.5 3.4/2.3 0.68/0.46 0.0161.2x/2.3x 0.4x — — Lymphoid depletion: thymus, lymph nodes 2  15/10.22.5/1.7 0.059  5.4x/10.2x 1.3x — ↑ Lymphoid fibrinogen depletion: ↑thymus, lymph globulins nodes, spleen, GALT 6 56/38 6.8/4.6 0.40820x/38x 9.1x BW ↓ Lymphoid loss lymphocytes depletion: (10% ↑ thymus,lymph vs. pre-study) neutrophils nodes, spleen, hypoactivity, ↑ GALTexcessive fibrinogen GI salivation, ↑ inflammation: increased globulinsesophagus, vomitus stomach, and colon, abnormal feces cecum Systemicinflammation: heart, aorta, meninges F_(u) dog = 0.68; F_(u) mouse =0.36 (F_(u) = unbound fraction in species plasma) *Exposure multiplesusing TGI60 from a 21-day KPL4 xenograft study; TGI60 @ 2.8 uM hr total,1.0 uM hr free **a-IgM stimulated CD69 expression (whole-blood) IC₅₀ =45 nM ⁺Findings related to inflammation and lymphoid organs (e)alpelisib (BYL-719) Exposure Exposure Exposure AUC0- Cmax Exposuremultiples Dose 24 hr, D 7 D 7 Cmin multiples* to CD69 mg/kg/day (μM hr,(μM, D 7 (total/free, IC₅₀** Clinical Clinical Histopa- QD total/free)total/free) (μM, total) AUC) (total, C_(min)) Signs Pathology⁺ thology⁺3 14/1.0 1.97/0.14 0.20 0.2x/0.2x 0.3x — — — 10 31.7/2.3  3.58/0.26 0.620.4x/0.4x 1.0x — ↑ GI fibrinogen inflammation: neutrophilic infiltrates,large intestine 30 160/11.5 20.6/1.5  1.25 2.2x/2.0x 2.0x ↓ ↑ Lymphoidappetite, neutrophils depletion: ~10% ↑ thymus, lymph ↓ BW monocytesnodes, GALT ↑ GI globulins inflammation: ↑ neutrophilic fibrinogeninfiltrates in large and small intestine; rectal ulceration F_(u) dog =0.072; F_(u) mouse = 0.082 (F_(u) = unbound fraction in species plasma)*Exposure multiples using TGI60 from a 21-day KPL4 xenograft study;TGI60 @ 72 uM hr (total), 5.9 uM hr (free) **a-IgM stimulated CD69expression (whole-blood) IC₅₀ = 613 nM ⁺Findings related to inflammationand lymphoid organs

Administration of Formula I Compounds

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 1 mg to about 1000mg of Formula I compound. A typical dose may be about 10 mg to about 300mg of the compound. A dose may be administered once a day (QID), twiceper day (BID), or more frequently, depending on the pharmacokinetic andpharmacodynamic properties, including absorption, distribution,metabolism, and excretion of the particular compound. In addition,toxicity factors may influence the dosage and administration regimen.When administered orally, the pill, capsule, or tablet may be ingesteddaily or less frequently for a specified period of time. The regimen maybe repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

Formula I compounds of the present invention are useful for treating ahuman or animal patient suffering from a disease or disorder arisingfrom abnormal cell growth, function or behavior associated with PI3Ksuch as cancer, may thus be treated by a method comprising theadministration thereto of a compound of the present invention as definedabove. A human or animal patient suffering from cancer may also betreated by a method comprising the administration thereto of a compoundof the present invention as defined above. The condition of the patientmay thereby be improved or ameliorated.

Methods of the invention also include treating cancer selected frombreast, ovary, cervix, prostate, testis, genitourinary tract, esophagus,larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma,lung, epidermoid carcinoma, large cell carcinoma, non-small cell lungcarcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, pancreatic, myeloid disorders, lymphoma, hairy cells, buccalcavity, naso-pharyngeal, pharynx, lip, tongue, mouth, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's, leukemia, bronchus, thyroid, liver and intrahepatic bileduct, hepatocellular, gastric, glioma/glioblastoma, endometrial,melanoma, kidney and renal pelvis, urinary bladder, uterine corpus,uterine cervix, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, lymphocytic leukemia, chronic lymphoid leukemia(CLL), myeloid leukemia, oral cavity and pharynx, non-Hodgkin lymphoma,melanoma, and villous colon adenoma.

Based on expression analysis, immunohistochemical analysis and on cellline profiling, malignancies of the colon, breast, cervix, stomach,lung, and multiple myeloma are most likely to respond to PI3K modulatorsor inhibitors.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment of mammals including humans, it is normally formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. According to this aspect of the invention there is provideda pharmaceutical composition comprising a compound of this invention inassociation with a pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16th edition, Osol, A. Ed.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the hyperproliferative disorder.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of compounds of Formula I may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI. Compressed tablets may be prepared by compressing in a suitablemachine the active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base. If desired, the aqueous phase of thecream base may include a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulfoxide and relatedanalogs. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it desirably comprises a mixture ofat least one emulsifier with a fat or an oil or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withadditional therapeutic agents for the treatment of a disease or disorderdescribed herein, such as inflammation or a hyperproliferative disorder(e.g., cancer). In certain embodiments, a compound of Formula I iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with an additional, second therapeutic compoundthat has anti-inflammatory or anti-hyperproliferative properties or thatis useful for treating an inflammation, immune-response disorder, orhyperproliferative disorder (e.g., cancer). The additional therapeuticmay be a Bcl-2 inhibitor, a JAK inhibitor, an anti-inflammatory agent,an immunomodulatory agent, chemotherapeutic agent, anapoptosis-enhancer, a neurotropic factor, an agent for treatingcardiovascular disease, an agent for treating liver disease, ananti-viral agent, an agent for treating blood disorders, an agent fortreating diabetes, and an agent for treating immunodeficiency disorders.The second therapeutic agent may be an NSAID anti-inflammatory agent.The second therapeutic agent may be a chemotherapeutic agent. The secondcompound of the pharmaceutical combination formulation or dosing regimenpreferably has complementary activities to the compound of Formula Isuch that they do not adversely affect each other. Such compounds aresuitably present in combination in amounts that are effective for thepurpose intended. In one embodiment, a composition of this inventioncomprises a compound of Formula I, or a stereoisomer, tautomer, solvate,metabolite, or pharmaceutically acceptable salt or prodrug thereof, incombination with a therapeutic agent such as an NSAID.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other therapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of therapy, a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, may be combined with othertherapeutic, hormonal or antibody agents such as those described herein,as well as combined with surgical therapy and radiotherapy. Combinationtherapies according to the present invention thus comprise theadministration of at least one compound of Formula I, or a stereoisomer,tautomer, solvate, metabolite, or pharmaceutically acceptable salt orprodrug thereof, and the use of at least one other cancer treatmentmethod. The amounts of the compound(s) of Formula I and the otherpharmaceutically active therapeutic agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Additional therapeutic agents employed in combination with a compound ofFormula I include 5-FU, docetaxel, eribulin, gemcitabine, cobimetinib,ipatasertib, paclitaxel, tamoxifen, fulvestrant, GDC-0810,dexamethasone, palbociclib, bevacizumab, pertuzumab, trastuzumabemtansine, trastuzumab and letrozole.

Metabolites of Compounds of Formula I

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound. Accordingly, the inventionincludes metabolites of compounds of Formula I, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof. The kit may further comprise a labelor package insert on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, for example, bottles, vials, syringes,blister pack, etc. The container may be formed from a variety ofmaterials such as glass or plastic. The container may hold a compound ofFormula I or a formulation thereof which is effective for treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a compound of Formula I. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formula I Compounds

Compounds of Formula I may be synthesized by synthetic routes thatinclude processes analogous to those well-known in the chemical arts,particularly in light of the description contained herein, and those forother heterocycles described in: Comprehensive Heterocyclic ChemistryII, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; LiebigsAnnalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database).

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing Formula I compoundsand necessary reagents and intermediates are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wileyand Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995) and subsequent editionsthereof.

The Examples provide exemplary methods for preparing Formula Icompounds. Those skilled in the art will appreciate that other syntheticroutes may be used to synthesize the Formula I compounds. Althoughspecific starting materials and reagents are depicted and discussed inthe Figures and Examples, other starting materials and reagents can beeasily substituted to provide a variety of derivatives and/or reactionconditions. In addition, many of the exemplary compounds prepared by thedescribed methods can be further modified in light of this disclosureusing conventional chemistry well known to those skilled in the art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

In the methods of preparing Formula I compounds, it may be advantageousto separate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified to the desired degree of homogeneity by thetechniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like. Selection of appropriate methodsof separation depends on the nature of the materials involved, such as,boiling point and molecular weight in distillation and sublimation,presence or absence of polar functional groups in chromatography,stability of materials in acidic and basic media in multiphaseextraction, and the like.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

Compounds of the invention were prepared as illustrated in generalSchemes 1 and 2.

As shown in Scheme 1, 4-bromo-2-hydroxybenzaldehyde 2 may be obtained byformylating commercially available 3-bromophenol. Heating 2 withoxaldehyde and ammonium hydroxide affords 3. The oxazepin ring may beformed by heating 3 with 1,2-dibromoethane. Bis iodination may beinduced by reaction with N-iodosuccinimide, and the 3-iodo groupselectively removed through treatment with ethyl magnesium bromide atreduced temperature, to afford 6.

As shown in Scheme 2, 6 may be coupled to an appropriately substitutedoxazolidin-2-one using copper catalysis to provide 7. Bromo intermediate7 may be coupled to appropriately substituted amino acids under coppercatalysis, followed by HATU-mediated amide coupling with ammoniumchloride to provide compounds 8.

EXAMPLES Abbreviations

-   -   DMSO Dimethyl sulfoxide    -   ESI Electrospray ionization    -   HPLC High pressure liquid chromatography    -   LCMS Liquid chromatography mass spectrometry    -   min Minutes    -   N Normal    -   NMR Nuclear magnetic resonance    -   R_(T) Retention time

LCMS Method A: Experiments performed on a Waters Micromass ZQ2000quadrupole mass spectrometer linked to a Waters Acquity UPLC system witha PDA UV detector. The spectrometer has an electrospray source operatingin positive and negative ion mode. This system uses an Acquity BEH C181.7 um 100×2.1 mm column, maintained at 40° C. or an Acquity BEH ShieldRP18 1.7 μm 100×2.1 mm column, maintained at 40° C. and a 0.4 mL/minuteflow rate. The initial solvent system was 95% water containing 0.1%formic acid (solvent A) and 5% acetonitrile containing 0.1% formic acid(solvent B) for the first 0.4 minute followed by a gradient up to 5%solvent A and 95% solvent B over the next 5.6 minutes. This wasmaintained for 0.8 minutes before returning to 95% solvent A and 5%solvent B over the next 0.2 minutes. Total run time was 8 minutes.

LCMS Method B: Experiments performed on an Agilent 1100 HPLC coupledwith Agilent MSD mass spectrometer using ESI as ionization source. TheLC separation was using a Phenomenex XB-C18, 1.7 mm, 50×2.1 mm columnwith a 0.4 mL/minute flow rate. Solvent A is water with 0.1% formic acidand solvent B is acetonitrile with 0.1% formic acid. The gradientconsisted with 2-98% solvent B over 7 minutes and hold 97% B for 1.5minutes following equilibration for 1.5 minutes. LC column temperatureis 40° C. UV absorbance was collected at 220 nm and 254 nm and mass specfull scan was applied to all experiments.

Example 101(S)-2-((2-((S)-4-(Difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)propanamide101 Step 1: 4-Bromo-2-hydroxybenzaldehyde

Into a 20 L 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed 3-bromophenol (1300 g, 7.51mol), dichloromagnesium (1078 g, 11.3 mol), triethylamine (3034 g, 30.0mol) and acetonitrile (7.8 L). The mixture was stirred for 30 minutes at40° C. To the mixture was added paraformaldehyde (676 g, 22.6 mol) at80° C. The resulting solution was stirred for 6 hours at 76° C. Thisreaction was repeated 5 times. The combined reaction mixtures werequenched by the addition of 12 L of aqueous hydrogen chloride (4 N). ThepH value of the solution was adjusted to 5 with concentrated aqueoushydrogen chloride (12 N). The resulting solution was extracted with 1×20L of ethyl acetate. The organic extracts were evaporated in vacuo. Theresidue was purified via flash chromatography on silica gel (eluted: 15%ethyl acetate in petroleum ether) to give crude product which was washedwith 2.4 L of methyl tert-butyl ether: hexane (1:4). The resultantsolids were collected by filtration to yield 7.0 kg (78%) of the titlecompound as a yellow solid.

Step 2: 5-Bromo-2-(1H-imidazol-2-yl)phenol

Into a 20 L 4-necked round-bottom flask was placed a solution of4-bromo-2-hydroxybenzaldehyde (700 g, 3.50 mol) in methanol (7.0 L) andoxaldehyde (40%) (2540 g, 17.5 mol) followed by the dropwise additionover 4 hours of aqueous ammonia (25-28%, 3500 g) with stirring andmaintaining the temperature below 40° C. The resulting solution wasstirred for 15 hours at 30-35° C. This reaction was repeated 9 times.The combined 9 reaction mixtures were evaporated in vacuo keeping thetemperature below 45° C. The residue was diluted with 100 L of ethylacetate with stirring for 30 minutes. The solids were filtered out andthe resulting solution was diluted with water. The aqueous phase wasextracted with 35 L of ethyl acetate. The organic extracts wereevaporated under vacuum and the residue was purified via flashchromatography on silica gel (solvent gradient: 5-75% ethyl acetate inpetroleum ether) to yield 2.4 kg (29%) of the title compound as a yellowsolid.

Step 3: 9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

Into a 20 L 4-necked round-bottom flask was placed a solution of5-bromo-2-(1H-imidazol-2-yl)phenol (1.4 kg, 5.86 mol) inN,N-dimethylformamide (14 L) and cesium carbonate (7.2 kg, 22.1 mol).The mixture was stirred for 20 minutes. To the reaction mixture wasadded 1,2-dibromoethane (4.1 kg, 21.8 mol). The resulting solution wasstirred for 4-12 hours at 85-90° C., cooled to 15° C., and filtered. Thefilter cake was washed with 3.0 L of ethyl acetate. The filtrate wasdiluted with 14 L of ethyl acetate. The combined organic extracts werewashed with brine (4×14 L), dried over anhydrous sodium sulfate,filtered and evaporated in vacuo to yield 1.1 kg (71%) of the titlecompound as a light yellow solid. LCMS (ESI): [M+H]⁺=265; ¹H NMR (400MHz, DMSO-d₆) δ 8.32 (d, J=8.4, 1H), 7.35-7.24 (m, 3H), 7.06 (s, 1H),4.47-4.42 (m, 4H).

Step 4:9-Bromo-2,3-diiodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

Into a 20 L 4-necked round-bottom flask was placed9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (2.5 kg, 9.43mol) and N,N-dimethylformamide (12.5 L) followed by the addition ofN-iodosuccinimide (6.0 kg, 26.7 mol) in several batches with stirring.The resulting solution was stirred for 12 hours at 60° C., cooled to 15°C. with a water/ice bath, diluted with 12.5 L of water/ice, andfiltered. The filtered solids were recrystallized from petroleum etherto yield 4.0 kg (82%) of the title compound as a yellow solid.

Step 5: 9-Bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

To a 20 L 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed9-bromo-2,3-diiodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (800g, 1.55 mol) and tetrahydrofuran (2.4 L) followed by the dropwiseaddition of ethyl magnesium bromide (1 N solution in ether, 1.7 L) withstirring at −20° C., over 3.5 hours. The reaction mixture was stirredfor 3 hours keeping the temperature at -15° C. using an ice/salt bath.The resultant mixture was quenched by the addition of 3.0 L of saturatedaqueous ammonium chloride, and extracted with ethyl acetate (2×8.0 L).The combined organic extracts were washed with brine (2×10 L), driedover anhydrous sodium sulfate, filtered and evaporated in vacuo. Thecrude residue was triturated with 8.0 L of ethyl acetate: petroleumether (1:5), filtered, and washed with petroleum ether to yield 501 g(83%) of the title compound as a brown solid. LCMS (ESI): [M+H]⁺=391; ¹HNMR (400 MHz, DMSO-d₆) δ 8.22 (d, J=8.7, 1H), 7.55 (s, 1H), 7.30-7.25(m, 2H), 4.45-4.41 (m, 4H).

Step 6: (R)-2,2-Dimethyl-[1,3]dioxolane-4-carbaldehyde

Sodium periodate (57.0 g, 270 mmol) was dissolved in hot water (115 mL)and silica (200 g, 60 Å 220-440 mesh, particle size 35-75 μm) was added.The mixture was stirred vigorously until a free flowing powder wasobtained. This was added to a solution of1,2:5,6-bis-O-(1-methylethylidene)-D-mannitol (50 g, 190 mmol) indichloromethane (1.0 L) and the reaction was stirred at room temperaturefor 1 hour. The resultant mixture was filtered through a pad of Na₂SO₄and the solids washed thoroughly with dichloromethane. The combinedorganic extracts were evaporated in vacuo to yield 37.2 g (75%) of thetitle compound as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 9.73 (d,J=1.9 Hz, 1H), 4.38 (ddd, J=7.4, 4.7, 1.9 Hz, 1H), 4.18 (dd, J=8.8, 7.4Hz, 1H), 4.10 (dd, J=8.8, 4.7 Hz, 1H), 1.49 (s, 3H), 1.43 (s, 3H).

Step 7: (R)-4-Difluoromethyl-2,2-dimethyl-[1,3]dioxolane

To a solution of (R)-2,2-dimethyl-[1,3]dioxolane-4-carbaldehyde (7.08 g,54 mmol) in dichloromethane (50 mL) cooled in a water bath was added,dropwise, diethylaminosulfur trifluoride (8.4 mL, 62.6 mmol) and thereaction mixture was stirred at room temperature for 3 hours. Theresultant mixture was added dropwise to a rapidly stirring, ice coldsaturated aqueous sodium bicarbonate solution. The mixture was furtherextracted with dichloromethane. The combined organic extracts werewashed with brine, dried over magnesium sulfate, filtered and evaporatedin vacuo to yield 6.58 g (79%) of the crude title compound as an orangeoil. ¹H NMR (400 MHz, CDCl₃) δ 5.69 (td, J=55.8, 4.9 Hz, 1H), 4.27-4.17(m, 1H), 4.16-4.03 (m, 2H), 1.46 (s, 3H), 1.38 (s, 3H).

Step 8: (R)-3-(tert-Butyldimethylsilanyloxy)-1,1-difluoropropan-2-ol

HCl in dioxane (4 N, 10.8 mL, 43.2 mmol) was added to a solution of(R)-4-difluoromethyl-2,2-dimethyl[1,3]dioxolane (6.58 g, 43.2 mmol) inmethanol (40 mL) and the reaction mixture was stirred at roomtemperature for 30 minutes. The resultant mixture was evaporated invacuo and azeotroped with acetonitrile. The residue was dissolved inN,N-dimethylformamide (10 mL) and tert-butyldimethylsilyl chloride (6.53g, 43.2 mmol), triethylamine (9.0 mL, 64.9 mmol) and4-(dimethylamino)pyridine (catalytic) were added. The reaction mixturewas stirred at room temperature for 1 hour. The resultant mixture waswashed with water and then extracted with dichloromethane. The combinedorganic extracts were washed with brine, dried over magnesium sulfate,filtered and evaporated in vacuo. The resultant crude residue waspurified via flash chromatography on silica gel (solvent gradient: 0-30%ethyl acetate in cyclohexane) to yield 3.43 g (35%) of the titlecompound as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 5.66 (td, J=56.4,4.6 Hz, 1H), 3.76-3.60 (m, 2H), 2.46 (d, J=6.4 Hz, 1H), 0.81 (s, 9H),0.00 (s, 6H).

Step 9: ((S)-2-Azido-3,3-difluoropropoxy)-tert-butyldimethylsilane

Trifluoromethanesulfonic anhydride (2.9 mL, 17.4 mmol) was addeddropwise to a solution of(R)-3-(tert-butyldimethylsilanyloxy)-1,1-difluoropropan-2-ol (3.43 g,15.1 mmol) and pyridine (2.0 mL, 24.2 mmol) in dichloromethane (50 mL)at −20° C. and the reaction mixture stirred at −20° C. for 20 minutesand then at 0° C. for 1 hour. The resultant mixture was diluted with 0.5N aqueous HCl and extracted with dichloromethane. The combined organicextracts were dried over magnesium sulfate and evaporated in vacuo. Thecrude residue was dissolved in N,N-dimethylformamide (10 mL), sodiumazide (2.96 g, 45.5 mmol) was added and the reaction mixture was stirredat room temperature for 2 hours. The resultant mixture was diluted withwater and extracted with ethyl acetate. The combined organic extractswere washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo to yield 4.50 g of the crude title compound. ¹H NMR(400 MHz, CDCl₃) δ 5.74 (td, J=55.4, 4.4 Hz, 1H), 3.81-3.71 (m, 2H),3.58-3.47 (m, 1H), 0.81 (s, 9H), 0.00 (s, 6H).

Step 10: (S′)-1-(tert-Butyl dimethylsilanyloxymethyl)-2,2-difluoroethylamine

Palladium hydroxide on carbon (200 mg, 20%) was added to a solution of((R)-2-azido-3,3-difluoropropoxy)-tert-butyldimethylsilane (4.50 g,crude, assume ˜15.1 mmol) in ethyl acetate (20 mL) and methanol (2.0 mL)and the reaction stirred under a balloon of hydrogen for 16 hours. Thereaction was filtered, fresh palladium hydroxide on carbon (400 mg, 20%)added and the reaction mixture was stirred under a balloon of hydrogenfor 16 hours. The resultant mixture was filtered and the filtrate wasevaporated in vacuo to yield 3.08 g (90%) of the crude title product asa colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.66 (td, J=57.0, 4.7 Hz,1H), 3.71-3.57 (m, 2H), 3.00-2.89 (m, 1H), 1.42 (br s, 2H), 0.82 (s,9H), 0.00 (s, 6H).

Step 11: (S)-4-Difluoromethyloxazolidin-2-one

HCl in dioxane (4 N, 5.0 mL, 20 mmol) was added to a solution of(R)-1-(tert-butyldimethylsilanyloxymethyl)-2,2-difluoroethylamine (Org.Lett., Vol. 9, No. 1, 2007, 41-44) (2.30 g, 10.3 mmol) in methanol (5.0mL) and the reaction mixture was stirred at room temperature for 2hours. The mixture was evaporated in vacuo and the resultant oil wastriturated with diethyl ether to give a solid which was dried in vacuo.The solid was dissolved in a mixture of toluene (20 mL) and KOH (2.50 g,44.6 mmol in 20 mL water) at 0° C. Phosgene (16.3 mL, 20% in toluene)was added dropwise, the cooling bath was removed and the reactionmixture was stirred for 1 hour. The mixture was evaporated in vacuo, theresultant residue was extracted with hot industrial methylated spiritsand the solid was collected by filtration. The filtrate was evaporatedin vacuo and the resultant residue was purified via flash chromatographyon silica gel (solvent gradient: 0-100% ethyl acetate in cyclohexane) toyield 830 mg (68%) of the title compound as an off-white solid.[α]_(D)=+10.1 (c=2.37, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 5.96 (br s,1H), 5.78 (td, J=55.3, 4.8 Hz, 1H), 4.54 (t, J=9.2 Hz, 1H), 4.42 (dd,J=9.6, 4.4 Hz, 1H), 4.17-4.06 (m, 1H).

Step 12:(S)-3-(9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(difluoromethyl)oxazolidin-2-one

A mixture of9-bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (250 mg,0.64 mmol), (S)-4-difluoromethyloxazolidin-2-one (88 mg, 0.64 mmol),trans-N,N′-dimethyl-1,2-cyclohexane diamine (36 mg, 0.26 mmol), cuprousiodide (24 mg, 0.13 mmol) and potassium carbonate (177 mg, 1.28 mmol) indioxane (3.0 mL) was degassed with argon under sonication. The reactionmixture was heated at 100° C. for 5 h and then allowed to cool to roomtemperature. The resultant mixture was diluted with 15% aqueous ammoniaand extracted with ethyl acetate. The combined organic extracts werewashed with brine, dried over magnesium sulfate, filtered and evaporatedin vacuo. The resultant residue was triturated with methanol and thenpurified via preparative HPLC [C18, 60% acetonitrile (0.1% formic acid)in water (0.1% formic acid), 20 minute run] to yield 20 mg (8%) of thetitle compound as a white solid. LCMS (ESI): [M+H]⁺=400/402. ¹H NMR (400MHz, CDCl₃) δ 8.19 (d, J=9.2 Hz, 1H), 7.29 (s, 1H), 7.24-7.19 (m, 2H),6.65 (ddd, J=57.8, 54.5, 1.0 Hz, 1H), 4.87 (ddd, J=24.0, 9.2, 4.0 Hz,1H), 4.73 (dd, J=9.5, 4.2 Hz, 1H), 4.53 (t, J=9.2 Hz, 1H), 4.48-4.43 (m,2H), 4.38-4.33 (m, 2H).

Step 13:(S)-2-((2-((S)-4-(Difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)propanamide

(S)-3-(9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(difluoromethyl)oxazolidin-2-one(600 mg, 1.50 mmol), L-alanine (267 mg, 3.00 mmol), cuprous iodide (57mg, 0.30 mmol) and potassium phosphate tribasic (637 mg, 3.00 mmol) weresuspended in dimethyl sulfoxide (6.0 mL). The reaction mixture washeated at 100° C. for 2 hours. Upon allowing to cool to roomtemperature, dimethyl sulfoxide (4.0 mL), ammonium chloride (480 mg,9.00 mmol), and triethylamine (3.1 mL, 22.5 mmol) were added. To theresultant stirred suspension was added,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (5.10 g, 13.5 mmol), portion-wise over 5minutes. The reaction mixture was stirred at room temperature for 1 hourand then filtered through Celite®, washing with ethyl acetate. Theorganic extracts were washed with saturated aqueous sodium bicarbonateand the aqueous phase was extracted with ethyl acetate. The combinedorganic extracts were washed with brine, dried over sodium sulfate,filtered and evaporated in vacuo. The crude residue was purified viaflash chromatography on silica gel (solvent gradient: 0-5% methanol indichloromethane) and then by chiral supercritical fluid chromatographyto yield 294 mg (46%) of 101 as an off-white solid. LCMS (ESI):R_(T)=2.89 [M+H]⁺=408, Method=A; ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (d,J=8.7 Hz, 1H), 7.38 (br s, 1H), 7.18 (s, 1H), 7.00 (br s, 1H), 6.71 (t,J=55.9 Hz, 1H), 6.41 (dd, J=8.8, 2.3 Hz, 1H), 6.16 (d, J=7.2 Hz, 1H),6.09 (d, J=1.9 Hz, 1H), 5.02-4.89 (m, 1H), 4.63-4.52 (m, 2H), 4.39-4.30(m, 4H), 3.76 (quintet, J=7.0 Hz, 1H), 1.30 (d, J=7.1 Hz, 3H).

Example 102(S)-2-cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide102 Step 1: (R)-4-Methyloxazolidin-2-one

To a mixture of D-alaninol (8.65 g, 0.12 mmol) in toluene and aqueousKOH (124 mL, 12.5% aq, 0.28 mmol) at 0° C. was added phosgene (72.7 mL,20% in toluene, 0.14 mmol) at such a rate that the internal temperatureremained <5° C. The reaction mixture was stirred at 0° C. for a further40 minutes then evaporated to dryness. The crude residue was extractedwith industrial methylate spirits, the slurry was filtered and thefiltrate evaporated in vacuo. The resultant residue was purified viaflash chromatography on silica gel (solvent gradient: 40-100% ethylacetate in cyclohexane) to yield 10.4 g (90%) of the title compound as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ 6.00 (br s, 1H), 4.50 (t, J=6.5Hz, 1H), 4.07-3.97 (m, 1H), 3.95 (dd, J=7.8, 6.2 Hz, 1H), 1.30 (d, J=6.1Hz, 3H).

Step 2:(R)-3-(9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-methyloxazolidin-2-oneand(R)-3-(9-Iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-methyloxazolidin-2-one

A mixture of9-bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (30.0 g,76.7 mmol), (R)-4-methyloxazolidin-2-one (7.70 g, 76.7 mmol), cuprousiodide (1.61 g, 8.40 mmol), trans-N,N′-dimethyl-1,2-cyclohexane diamine(2.7 mL, 16.9 mmol) and potassium carbonate (14.9 g, 107 mmol) weresuspended in 1,4-dioxane (200 mL) and the reaction mixture degassed withargon under sonication. The resultant mixture was heated at 100° C. for16 h. The reaction mixture was diluted with aqueous ammonia solution(˜16%) and extracted with ethyl acetate. The combined organic extractswere washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The resultant residue was purified via flashchromatography on silica gel (solvent gradient: 0-100% ethyl acetate incyclohexane) to yield 13.4 g (˜42%) of the title compounds (˜2:1 mixtureof 9-Br: 9-I products). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J=7.6 Hz,0.33H), 8.11 (d, J=6.9 Hz, 0.66H), 7.42-7.38 (m, 1H), 7.28-7.24 (m,1.33H), 7.23-7.18 (m, 0.66H), 4.77-4.68 (m, 1H), 4.58 (t, J=8.3 Hz, 1H),4.49-4.39 (m, 2H), 4.37-4.30 (m, 2H), 4.08 (dd, J=8.4, 4.5 Hz, 1H),1.57-1.50 (m, 3H).

Step 3:(R)-3-(9-Bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-2-yl)-4-methyl-oxazolidin-2-one

80 mg of a mixture of(R)-3-(9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-methyloxazolidin-2-oneand(R)-3-(9-Iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-methyloxazolidin-2-onewas separated via chiral SFC, to yield 27.6 mg of the title compound.LCMS (ESI): [M+H]⁺=364.0/366.0/367.2; ¹H NMR (400 MHz, DMSO-d₆) δ 8.22(d, J=8.7 Hz, 1H), 7.35 (s, 1H), 7.31 (dd, J=8.7, 2.1 Hz, 1H), 7.25 (d,J=2.0 Hz, 1H), 4.65-4.54 (m, 2H), 4.49-4.43 (m, 4H), 4.09-4.06 (m, 1H),1.42 (d, J=6.0 Hz).

Step 4: Methyl(S)-2-cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetate

A mixture of(4R)-3-(9-bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-2-yl)-4-methyl-oxazolidin-2-one(0.2746 mmol, 100 mg), cuprous iodide (0.084 mmol, 16 mg),(25)-2-amino-2-cyclobutyl-acetic acid (1.10 mmol, 142 mg) and potassiumphosphate tribasic (1.37 mmol, 297 mg) in dimethyl sulfoxide (3 mL) wereheated under microwave irradiation at 120° C. for 2 hours. The reactionwas cooled to room temperature, and iodomethane (1.4 mmol, 0.086 mL) wasadded and the reaction was extracted with dichloromethane and water. Thecombined organic extracts were combined, washed with brine, and driedwith sodium sulfate, filtered and evaporated in vacuo. The crude productwas purified via flash chromatography on silica gel (24 g silica,solvent gradient: 5-40% 3:1 isopropyl acetate:methanol indichloromethane) to yield 100 mg (85%) of the title compound.

Step 5:(S)-2-cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide

To a solution of methyl(2S)-2-cyclobutyl-2-[[2-[(4R)-4-methyl-2-oxo-oxazolidin-3-yl]-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-9-yl]amino]acetate(0.234 mmol, 100 mg) in tetrahydrofuran (5 mL) was added water (0.45 mL)and lithium hydroxide monohydrate (0.357 mmol, 15 mg). The reactionmixture was stirred at room temperature for 6 hours. The reactionmixture was evaporated in vacuo. To a solution of the resulting residuein N,N-dimethylformamide (3 mL) was added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (0.353 mmol, 137 mg), ammonium chloride(0.71 mmol, 38 mg) and N,N-diisopropylethylamine (0.705 mmol, 0.123 mL)and the reaction mixture stirred at room temperature for 1 hour. Thereaction mixture was evaporated in vacuo and the resultant residuetreated with water then extracted with dichloromethane. The combinedorganic extracts were washed with brine, dried over magnesium sulfateand evaporated in vacuo. The crude product was purified viareverse-phase HPLC, followed by SFC and lyophilized to yield 15.0 mg(15%) of 102. LCMS (ESI): R_(T) (min)=3.03, [M+H]⁺=412.2, method=D; ¹HNMR (400 MHz, DMSO-d₆) δ 7.96 (d, J=8.8 Hz, 1H), 7.39-7.36 (brs, 1H),7.13 (s, 1H), 7.00-6.97 (brs, 1H), 6.44 (dd, J=8.9, 2.3 Hz, 1H), 6.14(d, J=2.3 Hz, 1H), 5.96 (d, J=7.7 Hz, 1H), 4.62-4.49 (m, 2H), 4.38-4.28(m, 4H), 4.06-4.03 (m, 1H), 3.70-3.61 (m, 1H), 2.06-1.75 (m, 6H),1.42-1.34 (m, 3H).

Example 103(S)-2-Cyclopropyl-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide103

A mixture of(S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(difluoromethyl)oxazolidin-2-onefrom Example 101, step 12 (400 mg, 1.00 mmol), L-cyclopropylglycine (230mg, 2.00 mmol), cuprous iodide (38 mg, 0.20 mmol) and potassiumphosphate tribasic (424 mg, 2.00 mmol) in dimethyl sulfoxide (2.0 mL)were degassed with argon under sonication. The mixture was heated at100° C. for 5 hours then cooled to ambient temperature. The resultantmixture was diluted with dimethyl sulfoxide (5.0 mL) and ammoniumchloride (320 mg, 6.00 mmol) and triethylamine (1.4 mL, 10.0 mmol) wereadded. To the stirred suspension was then added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (2.28 g, 6.0 mmol), portion-wise, and thereaction mixture was stirred at room temperature for 10 minutes. Theresultant mixture was diluted with 15% aqueous ammonia solution andextracted with ethyl acetate. The combined organic extracts were washedwith brine, dried over magnesium sulfate, filtered and evaporated invacuo. The crude residue was purified via flash chromatography on silicagel (solvent gradient: 0-7% methanol in ethyl acetate). The residue wasdissolved in a minimum of acetonitrile. Water was then added toprecipitate a solid which was collected by filtration and dried in vacuoto yield 324 mg (75%) of 103 as an off-white solid. LCMS (ESI): R_(T)(min)=3.21, [M+H]⁺=434, Method=A; ¹H NMR (400 MHz, DMSO-d₆) δ 7.98 (d,J=8.6 Hz, 1H), 7.40 (br s, 1H), 7.17 (s, 1H), 7.03 (br s, 1H), 6.71 (t,J=56.0 Hz, 1H), 6.42 (dd, J=8.9, 2.4 Hz, 1H), 6.24 (d, J=7.2 Hz, 1H),6.09 (d, J=2.4 Hz, 1H), 5.01-4.89 (m, 1H), 4.63-4.51 (m, 2H), 4.38-4.29(m, 4H), 3.15 (t, J=7.7 Hz, 1H), 1.16-1.05 (m, 1H), 0.56-0.44 (m, 3H),0.33-0.25 (m, 1H).

Example 104(S)-2-Cyclopropyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide104

A mixture of(R)-3-(9-bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepin-2-yl)-4-methyl-oxazolidin-2-one(Example 102, step 3) (1.098 mmol, 400 mg), cuprous iodide (0.330 mmol,62.8 mg), (2S)-2-amino-2-cyclopropyl-acetic acid (3.295 mmol, 379.3 mg)and potassium phosphate tribasic (4.393 mmol, 951.5 mg) in dimethylsulfoxide (35 mmol, 2.5 mL) was heated at 110° C. for 2 hours undermicrowave irradiation. The reaction was cooled to room temperature. Tothe reaction mixture was added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (12.08 mmol, 4260 mg), ammonium chloride(12.08 mmol, 646 mg) and triethylamine (1.53 mL, 11.0 mmol). After 20minutes at room temperature, the reaction mixture was treated with waterthen extracted with dichloromethane. The combined organic extracts werewashed with brine, dried over magnesium sulfate, filtered and evaporatedin vacuo. The crude product was purified via reverse-phase HPLC andlyophilized to yield 110 mg (25% over 2 steps) of 104. LCMS (ESI): R_(T)(min)=2.588, [M+H]⁺=398.2, method=B; ¹H NMR (400 MHz, DMSO-d₆) δ 7.96(d, J=8.8 Hz, 1H), 7.39 (d, J=2.2 Hz, 1H), 7.13 (s, 1H), 7.02 (d, J=2.3Hz, 1H), 6.42 (dd, J=8.9, 2.4 Hz, 1H), 6.20 (d, J=7.1 Hz, 1H), 6.09 (d,J=2.4 Hz, 1H), 4.61-4.49 (m, 2H), 4.40-4.27 (m, 4H), 4.10-3.99 (m, 1H),3.22-3.09 (m, 1H), 1.42-1.36 (m, 3H), 1.16-1.04 (m, 1H), 0.56-0.42 (m,3H), 0.32-0.27 (m, 1H).

Example 105(S)-2-Cyclopropyl-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide105 Step 1: (R)-1-(tert-Butyldimethylsilanyloxy)-3-fluoropropan-2-ol

tert-Butyldimethylsilyl chloride (1.60 g, 10.63 mmol) was added to asolution of (R)-3-fluoropropane-1,2-diol (1.00 g, 10.6 mmol),triethylamine (1.93 mL, 13.8 mmol) and catalytic4-(dimethylamino)pyridine in dichloromethane at 0° C. and the reactionmixture was allowed to warm to room temperature and stirred at roomtemperature for 16 hours. The reaction mixture was diluted with waterand extracted with dichloromethane. The combined organic fractions werewashed with brine, dried over magnesium sulfate, filtered and evaporatedin vacuo. The resultant crude residue was purified via flashchromatography on silica gel (solvent gradient: 0-40% ethyl acetate incyclohexane) to yield 1.80 g (81%) of the title compound as a colorlessoil. ¹H NMR (400 MHz, CDCl₃) δ 4.45-4.36 (m, 1H), 4.34-4.25 (m, 1H),3.87-3.73 (m, 1H), 3.66-3.56 (m, 2H), 2.30 (d, J=6.0 Hz, 1H), 0.82 (s,9H), 0.00 (s, 6H).

Step 2: (S)-2-Azido-3-fluoropropoxy)-tert-butyldimethylsilane

Trifluoromethanesulfonic anhydride (1.67 mL, 9.93 mmol) was addeddropwise to a solution of(R)-1-(tert-butyldimethylsilanyloxy)-3-fluoropropan-2-ol (1.80 g, 8.60mmol) and pyridine (1.2 mL, 13.8 mmol) in dichloromethane at −20° C. andthe reaction mixture stirred at −20° C. for 20 minutes then at 0° C. for30 minutes. The reaction mixture was diluted with 0.5 N aqueous HCl andextracted with dichloromethane. The combined organic extracts were driedover magnesium sulfate, filtered and evaporated in vacuo. The residuewas dissolved in N,N-dimethylformamide (5.0 mL) and sodium azide (1.68g, 25.9 mmol) was added. The reaction mixture was stirred at roomtemperature for 2 hours. The resultant mixture was diluted with waterand extracted with ethyl acetate. The combined organic extracts werewashed with brine, dried over magnesium sulfate, filtered and evaporatedin vacuo to yield the crude title compound which was carried forwardwithout purification. ¹H NMR (400 MHz, CDCl₃) δ 4.58-4.26 (m, 2H),3.75-3.63 (m, 2H), 3.62-3.46 (m, 1H), 0.80 (s, 9H), 0.00 (s, 6H).

Step 3: (S)-1-(tert-Butyldimethylsilanyloxymethyl)-2-fluoroethylamine

Palladium hydroxide (400 mg, 20% on carbon) was added to a solution of((S)-2-azido-3-fluoropropoxy)-tert-butyldimethylsilane (crude, assume8.60 mmol) in ethyl acetate (15 mL) and methanol (5.0 mL) and thereaction mixture was stirred under a balloon of hydrogen for 16 hours.The resultant mixture was filtered, fresh palladium hydroxide (400 mg,20% on carbon) was added and the reaction was stirred under a balloon ofhydrogen for a further 16 hours. The resultant mixture was filtered andthe filtrate was evaporated in vacuo to yield the title compound as a˜2:1 mixture of product: starting material, which was carried forwardwithout purification.

Step 4: (S)-4-Fluoromethyloxazolidin-2-one

HCl in dioxane (4 N, 2.0 mL, 8.00 mmol) was added to a solution of(S)-1-(tert-butyldimethyl silanyloxymethyl)-2-fluoroethylamine (crude,assume 8.60 mmol) in methanol (3.0 mL) and the resulting mixture wasstirred at room temperature for 2 hours. The reaction mixture wasevaporated in vacuo. The resultant residue was dissolved in a mixture oftoluene (20 mL) and KOH (2.89 g, 51.6 mmol, 12.5% aq) at 0° C. To thismixture was added, dropwise, phosgene (13.6 mL, 20% in toluene), thecooling bath was removed and the resultant mixture was stirred for 1hour. The reaction mixture was evaporated in vacuo and the resultantresidue was extracted with hot industrial methylated spirits. Thefiltrate was evaporated in vacuo and the resultant residue was purifiedvia flash chromatography on silica gel (solvent gradient: 50-100% ethylacetate in cyclohexane) to yield 450 mg (44%, 3 steps) of the titlecompound as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 5.69 (br s,1H), 4.59-4.42 (m, 2H), 4.42-4.32 (m, 1H), 4.25-4.08 (m, 2H).

Step 5:(S)-3-(9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-oneand(S)-3-(9-Iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-one

A mixture of9-bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (722 mg,1.85 mmol), (S)-4-fluoromethyloxazolidin-2-one (220 mg, 1.85 mmol),3,4,7,8-tetramethyl-1,10-phenanthroline (131 mg, 0.55 mmol),Cu(OAc)₂.H₂O (74 mg, 0.37 mmol), potassium carbonate (510 mg, 3.70 mmol)and dioxane (6.0 ml) were sealed in a tube and the mixture degassed withargon under sonication. The reaction mixture was heated at 100° C. for72 hours. The resultant reaction mixture was diluted with 15% aqueousammonia and extracted with ethyl acetate. The combined organic extractswere washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The crude residue was purified via flashchromatography on silica gel (solvent gradient: 0-100% ethyl acetate incyclohexane) to yield 390 mg (53%) of the title compounds (approximate2:1 mixture of 9-Br and 9-I products). LCMS (ESI): [M+H]⁺=382/384/430;¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J=9.3 Hz, 0.7H), 8.05 (d, J=8.8 Hz,0.3H), 7.43-7.37 (m, 0.6H), 7.29 (s, 1.2H), 7.23-7.18 (m, 1.2H),5.03-4.66 (m, 3H), 4.60 (t, J=8.5 Hz, 1H), 4.54 (dd, J=8.6, 4.3 Hz, 1H),4.47-4.43 (m, 2H), 4.37-4.33 (m, 2H).

Step 6:(S)-2-Cyclopropyl-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide

A mixture of(S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-oneand(S)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-one(195 mg, ˜2:1 mix Br:I, ˜0.49 mmol), L-cyclopropylglycine (104 mg, 0.90mmol), cuprous iodide (17 mg, 0.09 mmol) and potassium phosphatetribasic (190 mg, 0.90 mmol) in dimethyl sulfoxide (1.5 mL) was degassedwith argon under sonication. The reaction mixture was heated at 100° C.for 16 hours then cooled to room temperature. The resultant mixture wasdiluted with dimethyl sulfoxide (1.0 mL) and ammonium chloride (144 mg,2.70 mmol) and triethylamine (950 μL, 6.75 mmol) were added. To thismixture was then added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (1.54 g, 4.05 mmol), portion-wise, and thereaction mixture was stirred at room temperature for 1 hour. Theresultant mixture was diluted with saturated aqueous sodium bicarbonatesolution and extracted with ethyl acetate. The combined organic extractswere washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The resultant crude residue was purified via flashchromatography on silica gel (solvent gradient: 0-5% methanol indichloromethane) and then further purified by flash chromatography onsilica gel (solvent gradient: 0-100% methyl acetate in cyclohexane) toyield 90 mg (48%) of 105 as an off-white solid. LCMS (ESI): R_(T)(min)=2.76 [M+H]⁺=416, Method=A; ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (d,J=8.8 Hz, 1H), 7.40 (br s, 1H), 7.17 (s, 1H), 7.03 (br s, 1H), 6.41 (dd,J=8.8, 2.3 Hz, 1H), 6.22 (d, J=7.1 Hz, 1H), 6.09 (d, J=2.2 Hz, 1H), 4.99(ddd, J=48.3, 9.8, 2.5 Hz, 1H), 4.81-4.56 (m, 3H), 4.40 (dd, J=8.6, 3.9Hz, 1H), 4.37-4.29 (m, 4H), 3.15 (t, J=7.6 Hz, 1H), 1.16-1.05 (m, 1H),0.54-0.43 (m, 3H), 0.33-0.25 (m, 1H).

Example 106(S)-2-((2-((S)-4-(Fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)propanamide106

A mixture of(S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-oneand(S)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(fluoromethyl)oxazolidin-2-one(Example 105, step 5) (195 mg, approximate 2:1 mixture 9-Br: 9-I,approximate 0.49 mmol), L-alanine (87 mg, 0.98 mmol), cuprous iodide (17mg, 0.09 mmol) and potassium phosphate tribasic (208 mg, 0.98 mmol) indimethyl sulfoxide (3.0 mL) were degassed with argon under sonication.The reaction mixture was heated at 100° C. for 4 hours then cooled toroom temperature. The resultant mixture was diluted with dimethylsulfoxide (3.0 mL) and ammonium chloride (157 mg, 2.94 mmol) andtriethylamine (683 μL, 4.8 mmol) were added. To this mixture was thenadded 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (1.10 g, 2.94 mmol), portion-wise, and thereaction mixture was stirred at room temperature for 30 minutes. Theresultant mixture was diluted with saturated aqueous sodium bicarbonatesolution and extracted with ethyl acetate. The combined organic extractswere washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The resultant residue was purified via flashchromatography on silica gel (solvent gradient: 0-5% methanol indichloromethane) and then further purified by chiral supercritical fluidchromatography to yield 36 mg (19%) of 106 as an off-white solid. LCMS(ESI): R_(T) (min)=2.43 [M+H]⁺=390, Method=A; ¹H NMR (400 MHz, DMSO-d₆)δ 7.96 (d, J=8.8 Hz, 1H), 7.37 (br s, 1H), 7.17 (s, 1H), 7.00 (br s,1H), 6.39 (dd, J=8.6, 1.6 Hz, 1H), 6.15 (d, J=7.0 Hz, 1H), 6.09 (d,J=1.6 Hz, 1H), 5.08-4.55 (m, 5H), 4.42-4.28 (m, 4H), 3.76 (quintet,J=7.2 Hz, 1H), 1.30 (d, J=7.2 Hz, 3H).

Example 107(S)-2-((2-((S)-4-(Difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)butanamide107

A mixture(S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-2-yl)-4-(difluoromethyl)oxazolidin-2-one(Example 101, step 12) (240 mg, 0.60 mmol), (S)-2-aminobutyric acid (124mg, 1.19 mmol), cuprous iodide (22.8 mg, 0.119 mmol), potassiumphosphate tribasic (255 mg, 1.19 mmol) and dimethyl sulfoxide (6.0 mL)was stirred under argon at 100° C. for 6 hours. The resultant mixturewas allowed to cool to room temperature and then ammonium chloride (188mg, 3.52 mmol) and triethylamine (1.2 mL, 8.80 mmol) were added. To thestirred suspension was added1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (2.01 g, 5.28 mmol), portion-wise, and thereaction mixture was stirred at room temperature for 1 hour. Theresultant mixture was diluted with ethyl acetate, washed with saturatedammonium chloride, dried over magnesium sulfate, filtered, andevaporated in vacuo. The crude product was purified via flashchromatography on silica gel (solvent gradient: 0-10% methanol in ethylacetate), further purified via reverse-phase HPLC, then chiralsupercritical fluid chromatography to yield 73.6 mg (30%) of 107 as awhite solid. LCMS (ESI): R_(T) (min)=3.13, [M+H]⁺=422, Method=A; ¹H NMR(400 MHz, DMSO-d₆) δ 7.99 (d, J=8.8 Hz, 1H), 7.40 (s, 1H), 7.17 (s, 1H),7.03 (s, 1H), 6.71 (t, J=56.0 Hz, 1H), 6.44 (dd, J=8.8, 2.2 Hz, 1H),6.13 (d, J=2.2 Hz, 1H), 6.09 (d, J=7.6 Hz, 1H), 5.02-4.89 (m, 1H),4.62-4.53 (m, 2H), 4.41-4.27 (m, 4H), 3.65-3.60 (m, 1H), 1.72-1.59 (m,2H), 0.94 (t, J=7.3 Hz, 3H).

Example 901 p110α (Alpha) PI3K Binding Assay

PI3K Binding assays are intended for determining the biochemical potencyof small molecule PI3K inhibitors. The PI3K lipid kinase reaction isperformed in the presence of PIP2:3PS lipid substrate (Promega #V1792)and ATP. Following the termination if the kinase reaction, turnover ofATP to ADP by the phosphorylation of the lipid substrate is detectedusing the Promega ADP-Glo™ (Promega #V1792) assay. Reactions are carriedout using the following conditions for each PI3K isoform as in Table 5.

TABLE 5 Reaction Final Kinase ATP PIP2:3PS Time Kinase SourceConcentration (uM) (uM) (min.) PI3K alpha Millipore 0.2 nM 40 50 120#14-602-K PI3K beta Promega 0.6 nM 40 50 120 #V1751 PI3K delta Millipore0.25 nM  40 50 120 #14-604-K PI3K gamma Millipore 0.4 nM 25 50 120#14-558-K

After 120 minutes of reaction time, the kinase reaction is terminated.Any ATP remaining after the reaction is depleted, leaving only ADP. Thenthe Kinase Detection Reagent is added to convert ADP to ATP, which isused in a coupled luciferin/luciferase reaction. The luminescent outputis measured and is correlated with kinase activity.

All reactions are carried out at room temperature. For each PI3K isoforma 3 μl mixture (1:1) of enzyme/lipid substrate solution is added to a384 well white assay plate (Perkin Elmer #6007299) containing 50 nl oftest compound or DMSO only for untreated controls. The reaction isstarted by the addition of 2 μl ATP/MgCl₂. The kinase reaction buffercontains 50 mM HEPES, 50 mM NaCl, 3 mM MgCl₂, 0.01% BSA, 1% DMSO, andenzyme and substrate concentrations as indicated in the above table. Thereaction is stopped by the addition of 10 μL ADP-Glo reagent. Plates areread in a Perkin Elmer Envision system using luminescence mode. 10 pointdose response curves are generated for each test compound. Ki values foreach compound are determined using the Morrison Equation.

Binding Assays: Initial polarization experiments were performed on anAnalyst HT 96-384 (Molecular Devices Corp, Sunnyvale, Calif.). Samplesfor fluorescence polarization affinity measurements were prepared byaddition of 1:3 serial dilutions of p110alpha PI3K (Upstate CellSignaling Solutions, Charlottesville, Va.) starting at a finalconcentration of 20 ug/mL in polarization buffer (10 mM Tris pH 7.5, 50mM NaCl, 4 mM MgCl₂, 0.05% Chaps, and 1 mM DTT) to 10 mM PIP₂(Echelon-Inc., Salt Lake City, Utah) final concentration. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume Proxiplates®(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the protein concentration. EC₅₀ values wereobtained by fitting the data to a four-parameter equation usingKaleidaGraph® software (Synergy software, Reading, Pa.). This experimentalso establishes the appropriate protein concentration to use insubsequent competition experiments with inhibitors.

Inhibitor IC₅₀ values were determined by addition of the 0.04 mg/mLp110alpha PI3K (final concentration) combined with PIP₂ (10 mM finalconcentration) to wells containing 1:3 serial dilutions of theantagonists in a final concentration of 25 mM ATP (Cell SignalingTechnology, Inc., Danvers, Mass.) in the polarization buffer. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume Proxiplates®(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the antagonist concentration, and the IC₅₀values were obtained by fitting the data to a 4-parameter equation inAssay Explorer software (MDL, San Ramon, Calif.).

Alternatively, inhibition of PI3K was determined in a radiometric assayusing purified, recombinant enzyme and ATP at a concentration of 1 μM(micromolar). The compound was serially diluted in 100% DMSO. The kinasereaction was incubated for 1 h at room temperature, and the reaction wasterminated by the addition of PBS. IC₅₀ values were subsequentlydetermined using sigmoidal dose-response curve fit (variable slope).

Example 902 Selective Inhibition of Mutant PI3Kα (Alpha)

The ability of a compound of the invention to act preferentially againstcells containing mutant PI3Kα (alpha) was determined by measuringinhibition of the PI3K pathway in SW48 isogenic cell lines: PI3Kαwild-type (parental), helical domain mutant E545K, and kinase domainmutant H1047R. The following assays are intended for determining thecellular potency and mutant selectivity of small molecule PI3Kαinhibitors. The assay utilizes isogenic cell lines that express PI3KαWT, PI3Kα mutant E545K/+(Horizon Discovery 103-001), or PI3Kα mutantH1047R/+ (Horizon Discovery 103-005). The potency of pPRAS40 inhibitionby PI3Kα in each cell line is measured after 24 hours of compoundtreatment. Mutant selectivity of PI3Kα inhibitors is determined by EC₅₀potency ratios in the WT vs. E545K cell lines and WT vs. H1047R celllines.

Cell Culture: Cell lines are maintained in a cell culture incubator at37° C. and 5% CO₂ in cell culture medium containing RPMI1640 (preparedat Genentech), 10% FBS (Gibco 16140-071), 2 mM L-glutamine (prepared atGenentech), and 10 mM HEPES pH 7.2 (prepared at Genentech). Cells aresplit every 72 hours at a ratio of 1:8 using 0.25% Trypsin-EDTA (Gibco25200).

Assay Procedure: Cells are harvested and plated in 384 well tissueculture treated assay plates (Greiner cat #781091) and incubatedovernight at 37° C. at 5% CO₂. The three cell lines (WT, E545K, andH1047R) are plated and assayed in parallel. The following day, testcompounds are serially diluted in dimethyl sulfoxide (DMSO) and added tocells (final DMSO concentration 0.5%). Cells are then incubated for 24hours at 37° C. and 5% CO₂. After 24 hours, cells are lysed and pPRAS40levels are measured using the Meso-Scale custom pPRAS40 384w Assay Kit(Meso Scale Discovery, cat # L21CA-1). Cell lysates are added to assayplates pre-coated with antibodies against phosphorylated PRAS40.Phosphorylated PRAS40 in samples are allowed to bind to the captureantibodies overnight at 4° C. The detection antibody (anti-total PRAS40,labeled with an electrochemiluminescent SULFO-TAG) is added to the boundlysate and incubated for 1 hour at room temperature. The MSD Read Bufferis added such that when a voltage is applied to the plate electrodes,the labels bound to the electrode surface emit light. The MSD SectorInstrument measures the intensity of the light, and quantitativelymeasures the amount of phosphor-PRAS40 in the sample. Percent inhibitionof PRAS40 phosphorylation by varying concentrations of test compounds iscalculated relative to untreated controls. EC₅₀ values are calculatedusing the 4 parameter logistic nonlinear regression dose-response model.

Statistical Analysis: EC₅₀ values represent the geometric mean of aminimum of 4 independent experiments. All statistics were performedusing KaleidaGraph Software (version 4.1.3). A Student t-Test wasperformed using unpaired data with equal variance to compare activityagainst mutant cells and wild-type cells. P<0.05 is considered to besignificant.

Example 903 In Vitro Cell Viability Assays

Cells were seeded (1,500 per well) in 384-well plates for 16 h. On day2, nine serial 1:3 compound dilutions were made in DMSO in a 96-wellplate. The compounds were then further diluted into growth media using aRapidplate robot (Zymark Corp.). The diluted compounds were then addedto quadruplicate wells in the 384-well cell plates and incubated at 37°C. and 5% CO₂. After 4 d, relative numbers of viable cells were measuredby luminescence using Cell Titer-Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(Perkin-Elmer). EC50 values were calculated using Prism 6.0 software(GraphPad).

Example 904 In Vivo Mouse Tumor Xenograft Efficacy

Mice:

Female severe combined immunodeficiency mice (C.B-17 SCID.bg CharlesRiver Labs, San Diego), NOD.SCID (Charles River Labs, Hollister) orNCR.nude mice (Taconic) were 8 to 9 weeks old and had a BW range of18-26 grams on Day 0 of the study. The animals received ad libitum waterand Laboratory Autoclavable Rodent Diet 5010 (LabDiet St. Louis, Mo.).The mice were housed in microisolators on a 12-hour light cycle.Genentech specifically complies with the recommendations of the Guidefor Care and Use of Laboratory Animals with respect to restraint,husbandry, surgical procedures, feed and fluid regulation, andveterinary care. The animal care and use program at Genentech isaccredited by the Association for Assessment and Accreditation ofLaboratory Animal Care International (AAALAC), which ensures compliancewith accepted standards for the care and use of laboratory animals. Themice were housed at Genentech in standard rodent micro-isolator cagesand were acclimated to study conditions for at least 3 days before tumorcell implantation. Only animals that appeared to be healthy and thatwere free of obvious abnormalities were used for the study

Tumor Implantation:

Xenografts were initiated with either cancer cells (HCC1954x1 or KPL4)or passage tumors (HCl-003). Cells were cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mpenicillin, 100 μg/mL (micrograms per ml) streptomycin sulfate and 25μg/mL gentamicin, harvested in the log-phase growth and resuspended in50% phenol red-free Matrigel (Becton Dickinson Bioscience; San Jose,Calif.) and Hank's Balanced Salt Solution at a concentration of 3×106 or5×106 cells/mL depending on the doubling time of the cell line. For theHCl-003 patient-derived model, 30 mg beeswax pellets containingapproximately 1 mg of 17β (beta)-estradiol were implanted subcutaneously3 days prior to implantation of tumor fragments. Tumor cells orfragments were implanted into the ⅔ mammary fat pad, and tumor growthwas monitored as the average size approached the target range of 100 to250 mm3. Once the majority of tumors reached the target range, Mice weredistributed into groups of 7-10 mice based on tumor volume.

Therapeutic Agents:

PI3K compounds were supplied as a free base in dry powder and stored atroom temperature protected from light. The vehicle for taselisib(GDC-0032) and BYL719 was 0.5% methylcellulose: 0.2% Tween 80 (MCT) indeionized water. The vehicle control for compound 101 was 0.5%methylcellulose/0.2% Tween-80 (MCT) nanosuspension. MCT nanosuspensionis prepared by initially preparing a MCT suspension. Once prepared, 1 mmglass beads and a rare earth magnetic stir bar are used to mill the MCTsuspension for about 24 hours into a fine nanosuspension. Particle sizeanalyzer was used to check final particle size. Drug doses were preparedweekly and stored at 4 C.

Treatment:

Mice were given (Vehicle) or stated mg/kg dosage of PI3K compounds(expressed as free-base equivalent), PO by gavage daily for 21-28 daysin a volume of 100 μL, microliters (5 mL/kg).

Endpoint:

Tumor volume was measured in 2 dimensions (length and width), usingUltra Cal 0 calipers (Model 54 10 111; Fred V. Fowler Company), asfollows: tumor volume (mm³)=(length×width²)×0.5 and analyzed using Excelversion 11.2 (Microsoft Corporation). A linear mixed effect (LME)modeling approach was used to analyze the repeated measurement of tumorvolumes from the same animals over time (Pinheiro J, et u|. nlme: linearand nonlinear mixed effects models, 2009; R package version 325. Thisapproach addresses both repeated measurements and modest dropouts due toany non-treatment-related death of animals before study end. Cubicregression splines were used to tit a nonlinear profile to the timecourses of log 2 tumor volume at each dose level. These nonlinearprofiles were then related to dose within the mixed model. Tumor growthinhibition as a percentage of vehicle control (% TGI) was calculated asthe percentage of the area under the fitted curve (AUC) for therespective dose group per day in relation to the vehicle, using thefollowing formula: % TGI=100×(1−AUC_(dose)/AUC_(veh)). Using thisformula, a TGI value of 100% indicates tumor stasis, a TGI value of morethan (>) 1% but less than (<) 100% indicates tumor growth delay, and aTGI value of more than (>) 100% indicates tumor regression. Partialresponse (PR) for an animal was defined as a tumor regression of morethan (>) 50% but less than (<) 100% of the starting tumor volume.Complete response (CR) was defined as 100% tumor regression (i.e., nomeasurable tumor) on any day during the study.

Toxicity:

Animals were weighed daily for the first five days of the study andtwice weekly thereafter. Animal body weights were measured using anAdventurer Pro® AV812 scale (Ohaus Corporation). Percent weight changewas calculated as follows: body weight change(%)=[(weight_(day new)−weight_(day 0))/weight_(day 0)]×100. The micewere observed frequently for overt signs of any adverse,treatment-related side effects, and clinical signs of toxicity wererecorded when observed. Acceptable toxicity is defined as a group meanbody weight (BW) loss of less than 20% during the study and not morethan one treatment-related (TR) death among ten treated animals. Anydosing regimen that results in greater toxicity is considered above themaximum tolerated dose (MTD). A death is classified as TR ifattributable to treatment side effects as evidenced by clinical signsand/or necropsy, or may also be classified as TR if due to unknowncauses during the dosing period or within 10 days of the last dose, Adeath is classified as NTR if there is no evidence that death wasrelated to treatment side effects.

Example 905 Cell Culture and In Vitro Inhibitor Experiments

Cell lines were grown under standard tissue culture conditions in RPMImedia with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mLstreptomycin. HCC-1954 and HDQ-P1 are breast cancer cell lines (AmericanType Culture Collection; Manassas, Va. HCC-1954 and HDQ-P1 cells wereplaced in each well of a 6-well tissue culture plate at 800,00cells/well and incubated at 37° C. overnight. Cells were incubated withthe indicated concentrations of each compound for 24 hours. Followingincubation, cells were washed once with cold phosphate-buffered saline(PBS) and lysed in Biosource™ Cell Extraction Buffer (Invitrogen;Carlsbad, Calif.) supplemented with protease inhibitors (F.Hoffman-LaRoche; Mannheim, Germany), 1 mM phenylmethylsulfonyl fluoride,and Phosphatase Inhibitor Cocktails 1 and 2 (Sigma-Aldrich; St. Louis,Mo.). Protein concentrations were determined using the Pierce BCAProtein Assay Kit (Thermo Fisher Scientific; Rockford, Ill.).

Protein Assays

Protein concentration was determined using the Pierce BCA Protein AssayKit (Rockford, Ill.). For immunoblots, equal protein amounts wereseparated by electrophoresis through NuPage Bis-Tris 4-12% gradient gels(Invitrogen; Carlsbad, Calif.); proteins were transferred ontoNitrocellulose membranes using the IBlot system and protocol fromInVitrogen. Antibodies to p110alpha, and phospho-Akt (Ser473), wereobtained from Cell Signaling (Danvers, Mass.). Antibodies to □-actin andGAPDH were from Sigma.

Example 906 B-Cell CD69 Expression, an Whole Blood Assay for CD69Expression in CD19⁺ CD27⁻B Cells

Cell Culture:

Human whole blood was dispensed into 96-deep well plates at 100 μl perwell. Compounds were diluted in DMSO to generate the desired stockconcentrations and then further diluted in PBS to the desired workingconcentration and added in a volume of 5.5 μL per well. Samples werethen incubated for 1 hour at 37° C. under 5% CO₂ before the addition of5 μg (10 μl per well) of Goat anti IgM. F(ab′)2 (Southern Biotech, AL),and incubated for 18 hours at 37° C. under 5% CO₂. All treatments weretested in duplicate.

Cell Isolation and Staining Procedures:

After incubation, the level of CD69 expression on CD19⁺ CD27⁻ cells wasdetermined by staining the whole blood samples with a cocktail of CD27;10 μl/well (clone L128; BD Biosciences, NJ) CD19; 7.5 μl/well (cloneSJ25C1; BD Biosciences, NJ) and CD69; 10 μl (clone FN50; BD Biosciences,NJ). In addition, human whole blood from each donor was stained withisotype-matched fluorescent control antibodies. After the addition ofthe appropriate antibody cocktail the whole blood samples were stainedfor 30 minutes in the dark and then lysed using BD Pharm Lysis (BDBioscience, NJ). The resulting samples were then washed with FACS Buffer(Phosphate Buffered Saline (Ca/Mg++ free), 1 mM EDTA, 25 mM HEPES pH7.0, 1% Fetal Bovine Serum (Heat-Inactivated) and fixed in FACS Buffersupplemented with 0.1% Formaldehyde (Polysciences Inc, PA) and 0.1%Pluronic F-68 (Sigma, Mo.). Data were acquired using a BD LSR-II (BDBiosciences) with BD FACSDiva software.

CD69 Expression of CD19⁺ CD27⁻ B Cells.

Cells were assessed by flow cytometry for levels of CD19, CD27 and CD69using BD FACSDiva Software and the CD69 MH-Mean of the CD19⁺ CD27⁻lymphocyte population was determined. The concentration of compoundresulting in 50% inhibition of CD69 MFI-Mean (IC₅₀) was determined usingGenedata software (Genedata Screener, MA).

Example 907 HCC1954 and HDQP1 pPRAS40 EC₅₀

Cells are plated in 384 well tissue culture treated assay plates andincubated overnight. The following day, cells are treated with compoundsand incubated for 24 hours. After 24 hours, cells are lysed and pPRAS40levels are measured using the Meso-Scale assay platform. These celllines are quite useful for characterizing the selectivity of PI3Kαinhibitors for mutant PI3Kα. The HCC1954 cell line expresses mutantPI3Kα (E545K) vs WT in HDQP1.

Assay Principle:

The MSD platform provides a method of measuring the phosphorylatedlevels of pPRAS40 in a single sample. Cell lysates are added to assayplates pre-coated with antibodies against total PRAS40. Following celllysis, PRAS40 in samples is allowed to bind to the capture antibodies.The detection antibody (anti-phospho PRAS40), labeled with anelectrochemiluminescent compound MSD SULFO-TAG, is added to the boundlysate. The MSD Read Buffer is added such that when a voltage is appliedto the plate electrodes, the labels bound to the electrode surface emitlight. The MSD Sector Instrument measures the intensity of the light,and quantitatively measures the amount of phosphor EGFR in the sample(Meso Scale Assay Principle).

Materials:

Assay plate Black u-Clear bottom 384 well sterile, TC treated plates(Greiner cat #781091) Cell Types WT Parental Line: HDQ-P1 (CL131963)E545K Mutant: HCC1954 (CL130216) Media RPMI 1640 10% FBS (Gibco16140-071) 2 mM L-Glutamine (GNE in-house) 1% HEPES (GNE in-house) Othercell culture 0.25% Trypsin EDTA 1X (Invitrogen Gibco, cat # reagents25200) Assay reagent kit Whole Cell Lysate Kit custom pPRAS40 384w AssayKit (Meso Scale Discovery, cat # L21CA-1 for 100 plates)

Procedure:

-   -   Compounds prepared at concentration of 2 mM in DMSO. Prepare        DMSO compound titration plate, 1:3 in neat DMSO.    -   DMSO Mother plate contains 72 μl of 13 compounds.    -   Mutant selective control compound: Add 72 ul of 2 mM control        compound to well B2 on each assay plate. This control compound        demonstrates approximately 20 fold greater potency in the        HCC1954 cell line vs. the HDQP1 line.    -   Using a multi-channel pipette, transfer 36 ul from each compound        well to the well directly below (example B2 to C2) in order to        set up duplicate dose-response curves.    -   Use Biomek Fx method titled “SLS_serial dilution/1        plate_384_3_13_3×” to make serial dilutions of compounds in        mother plate.    -   Seal and keep both the DMSO Mother and Daughter plates with a        heat sealer when not in use.

Day 1: Cell Plating

-   -   1. Seed 12,500 cells in 45 ul medium for each cell line. Allow        cells to settle/attach to plate for 15-20 min at room        temperature.    -   2. Incubate cells overnight in a 37° C. humidity and CO2        controlled incubator.

Day 2: Compound Plate Preparation and Compound Treatment

-   -   1. For the 10× intermediate dilution plate: add 95 ul serum-free        media into a standard profile greiner 384 well polypropylene        plate.    -   2. Use biomek Fx protocol for intermediate compound dilution in        media and addition to cells: “SLS Intermed Dil Add 5 ul to Cells        Jul. 13 2012.” This Biomek protocol transfers 5 ul from the DMSO        daughter plate to the intermediate dilution plate containing 95        ul medium and mixes the media+compounds. The method then        transfers 5 ul from the intermediate dilution plate to the        appropriate cell plate.    -   3. Incubate the treated cells at 37 degrees for 24 hours in a 5%        CO2 humidified incubator.

Day 3: Cell Lysis and Addition to MSD Plates

Block the MSD assay plate with 50 ul 3% Blocker A/1×MSD Wash Buffer 1-2hr at room temp. This solution can be stored at 4° C. for up to onemonth. Blocking buffer A contains 1×MSD wash buffer. 20 mL 1× Tris Washbuffer and 600 mg Blocker A.

Prepare Lysis Buffer:

3 plates 6 plates 9 plates 12 plates (mL) (mL) (mL) (mL) MSD LysisBuffer 65.0 120 180 240 Phosphatase Inh. Cocktail 1 0.65 1.2 1.8 2.4 (orSigma phosphatase Inhib 2 Cat# P5726-5 mL) Phosphatase Inh. Cocktail 20.65 1.2 1.8 2.4 (or Sigma phosphatase Inhib 3, Cat# P0044-5 mL)Protease inh 0.65 1.2 1.8 2.4 (or Sigma Cat# P2714-1BTL resuspended in10 mL PBS)

Aspirate Media and Lyse the Cells

-   -   1. Lyse the cells in 50 μl lysis buffer. Lyse at room        temperature for 10-20 minutes on plate shaker.    -   2. While cells are lysing, wash blocked plates 1×MSD wash        buffer.    -   3. Transfer 42 ul lysates (21+21 μL) to a blocked MSD pPRAS40        assay plate.    -   4. Seal MSD plates and incubate at 4° C. with shaking overnight.

Day 4: MSD Assay/Detection

-   -   8. Make a solution of 1% Blocker A in 1×MSD wash buffer. (20 mL        1× Tris Wash buffer and 200 mg Blocker A (1% w/v). This solution        can be stored at 4° C. for up to one month.    -   9. Wash the MSD plates with 1×MSD wash buffer.    -   10. Add 10 μl of the diluted SULFO-TAG detection antibody to the        plates. Incubate for 1 hr with shaking at room temp.

3 plates 6 plates 9 plates 12 plates (mL) (mL) (mL) (mL) 1% blocker A in13.0 25.0 36.0 48.0 MSD wash buffer 2% blocker DM 0.13 0.25 0.36 0.48(100X) Sulfo-TAG anti- 0.26 0.5 0.72 0.96 pPRAS40 (50X)

-   -   11. Wash the plates 4× with 1×MSD wash buffer.    -   12. Add 35 μl 1× Read buffer with reverse pipetting to avoid        bubbles.    -   13. Read the plate immediately on the MSD SECTOR instrument.

Example 908 Co-Crystallography with p110α (Alpha)

N-terminally truncated p110α (alpha) was produced according to Chen etal and Nacht et al. (Chen, P., Y. L. Deng, S. Bergqvist, M. D. Falk, W.Liu, S. Timofeevski and A. Brooun “Engineering of an isolated p110alphasubunit of PI3Kalpha permits crystallization and provides a platform forstructure-based drug design,” (2014) Protein Sci 23(10): 1332-1340;Nacht, M. et al (2013) “Discovery of a potent and isoform-selectivetargeted covalent inhibitor of the lipid kinase PI3Kalpha,” J. Med.Chem. 56(3): 712-721).

Standard protocols were applied to production of crystals in thepresence of project compounds. Harvested crystals were preserved fordiffraction data collection by immersion in liquid nitrogen and mountedon a synchrotron beamline producing monochromatic X-rays. Diffractiondata were collected, reduced and merged using standard protocols. Thecrystallographic unit cells and space group were isomorphous with thosereported previously (Nacht, 2013; Chen, 2014). Placement of projectcompounds into electron density maps and crystallographic refinement toresolution limits of between 2.36 and 2.56 Å were performed usingstandard protocols.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

We claim:
 1. A compound selected from Formula I:

and stereoisomers, geometric isomers, tautomers, and pharmaceuticallyacceptable salts thereof, wherein: R¹ is selected from —CH₃, —CH₂CH₃,cyclopropyl, and cyclobutyl; R² is selected from —CH₃, —CHF₂, —CH₂F, and—CF₃.
 2. The compound of claim 1 wherein R¹ is —CH₃ or cyclopropyl. 3.The compound of claim 1 wherein R² is —CHF₂.
 4. The compound of claim 1wherein Formula I is:(S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)propanamide.5. The compound of claim 1 wherein Formula I is:(S)-2-cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide.6. The compound of claim 1 wherein Formula I is:(S)-2-cyclopropyl-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide.7. The compound of claim 1 wherein Formula I is:(S)-2-cyclopropyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide.8. The compound of claim 1 wherein Formula I is:(S)-2-cyclopropyl-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)acetamide.9. The compound of claim 1 wherein Formula I is:(S)-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)propanamide.10. The compound of claim 1 wherein Formula I is:(S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)amino)butanamide.11. A pharmaceutical composition comprised of a compound of claim 1 anda pharmaceutically acceptable carrier, glidant, diluent, or excipient.12. The pharmaceutical composition of claim 11 wherein thepharmaceutically acceptable carrier, glidant, diluent, or excipient isselected from silicon dioxide, powdered cellulose, microcrystallinecellulose, metallic stearates, sodium aluminosilicate, sodium benzoate,calcium carbonate, calcium silicate, corn starch, magnesium carbonate,asbestos free talc, stearowet C, starch, starch 1500, magnesium laurylsulfate, magnesium oxide, and combinations thereof.
 13. A process formaking a pharmaceutical composition comprising combining a compound ofclaim 1 with a pharmaceutically acceptable carrier, glidant, diluent, orexcipient.
 14. A method of treating cancer in a patient having cancercomprising administering to said patient a therapeutically effectiveamount of a compound of claim 1 wherein the cancer is selected frombreast cancer and non-small cell lung cancer.
 15. The method of claim 14further comprising administering to the patient an additionaltherapeutic agent selected from 5-FU, docetaxel, eribulin, gemcitabine,cobimetinib, ipatasertib, paclitaxel, tamoxifen, fulvestrant, GDC-0810,dexamethasone, palbociclib, bevacizumab, pertuzumab, trastuzumabemtansine, trastuzumab and letrozole.
 16. The method of claim 14 whereinthe cancer is breast cancer.
 17. The use of claim 16 wherein the breastcancer is estrogen receptor positive (ER+) breast cancer.
 18. The methodof claim 16 wherein the breast cancer subtype is Basal or Luminal. 19.The method of claim 14 wherein the cancer expresses a PIK3CA mutantselected from E542K, E545K, Q546R, H1047L and H1047R.
 20. The method ofclaim 14 wherein the cancer expresses a PTEN mutant.
 21. The method ofclaim 16 wherein the cancer is HER2 positive.
 22. The method of claim 16wherein the patient is HER2 negative, ER (estrogen receptor) negative,and PR (progesterone receptor) negative.
 23. A kit for the therapeutictreatment of breast cancer, comprising: a) the pharmaceuticalcomposition of claim 11; and b) instructions for use in the therapeutictreatment of breast cancer.